Antibodies to protein, FAF1

ABSTRACT

The present invention identifies a novel, Fas-associated factor 1 termed FAF1 which potentiates Fas-induced cell killing. The invention provides FAF1 nucleic acid and polypeptide compositions as well as methods of using these compositions in the therapeutic treatment of diseases resulting from dysregulation in apoptosis. Also provided are cells carrying and expressing the nucleic acid compositions and methods of using these cells to screen for agonists and antagonists of Fas-mediated apoptosis. Methods of isolating FAF1-interacting proteins are disclosed. Also provided are antibodies that bind FAF1, a hybridoma and a kit comprising the antibodies.

This invention was made with Government support under Grant No.HL-32898, awarded by the National Institutes of Health. The Governmenthas certain rights in this invention.

The present application is a Rule 60 Divisional Application of U.S.patent application Ser. No. 08/477,476, filed on June 7, 1995 now U.S.Pat. No. 5,750,653.

BACKGROUND OF THE INVENTION

Apoptosis or programmed cell death is an important physiological processin multicellular organisms, both during development and for homeostasis.Apoptosis allows the elimination of cells that are no longer necessary,that are produced in excess, that have developed improperly or that havesustained genetic damage. Apoptosis occurs in many different tissuesystems and must be properly regulated to maximize benefit to theindividual; when the mechanism is dysregulated, it may cause significantdisease. Both inhibition of cell death and inappropriate cell death maybe deleterious. For example, inhibition of cell death may contribute todisease in the immune system to allow persistence of self-reactive B andT cells, thus promoting autoimmune disease (Watanabe-Fukunaga et al.,Nature, 356:314-317 (1992)). Most importantly, cancer may result whencells that fail to die undergo further mutations, leading to atransformed state (Korsmeyer, S. J., Blood, 80:879-886 (1992)).

The protein, Fas, mediates apoptosis. A cell surface receptor, Fas playsan important role in the development and function of the immune system.Malfunction of the Fas system causes lymphoproliferative disorders andaccelerates autoimmune diseases. Exacerbation of Fas-mediated apoptosismay cause tissue destruction.

SUMMARY OF THE INVENTION

The present invention provides the identification and isolation of anovel Fas-associated factor 1, termed FAF1, and the FAF1-encoding DNA.As a cytoplasmic protein, FAF1 was shown herein to bind to the wild typebut not the inactive point mutant of Fas. FAF1 specifically interactswith the cytoplasmic domain of wild type Fas and potentiates Fas-inducedcell killing. FAF1 is a signal transducing molecule in the regulation ofapoptosis.

The FAF1 nucleic acids and polypeptides find many uses. It would bedesirable to be able to control apoptosis by enhancing or decreasing thesusceptibility of individual cell types to undergo apoptosis, especiallywhen dysregulation of the process leads to disease. In particular, itwould be desirable to provide therapeutic intervention in diseaseconditions where apoptosis is dysregulated due to Fas or FAF1malfunction. Blockage or activation of FAF1 function in apoptosis can beused for example, in the treatment of cancer, immune disorders such asautoimmune diseases, infectious diseases, and myocardial infarction andneuronal infarction in cardiovascular diseases. Heretofore, suchtherapeutic approaches involving FAF1 were not possible. The presentdiscovery of FAF1 fulfills these and other needs.

The present invention provides the nucleotide and amino acid sequence ofFAF1 as well as polypeptide and nucleic acid compositions based on FAF1.The uses and methods of use of the FAF1 nucleic acid and polypeptidecompositions are disclosed.

One aspect of this invention is to provide an isolated nucleic acidcomprising at least 85% sequence identity with the nucleotide sequenceof SEQ ID NO:1, an allelic or species variation thereof, or a fragmentthereof. A nucleic acid comprising the nucleotide sequence of SEQ IDNO:1 is provided. Also provided is a recombinant DNA molecule comprisingthe nucleotide sequence of SEQ ID NO:1 or a fragment thereof. In oneembodiment, the recombinant DNA molecule encodes a FAF1-GAL4transactivation domain fusion protein. A cell is provided which containsthe recombinant DNA molecule comprising the nucleotide sequence of SEQID NO:1 or a fragment thereof.

Another aspect of the invention is to provide an isolated polypeptidecomprising at least 85% sequence identity with the sequence of SEQ IDNO:2, an allelic or species variation thereof, or a fragment thereof.The polypeptide can be provided in a kit. The polypeptide may furthercomprise an influenza virus HA epitope tag. An isolated polypeptidecomprising the sequence of SEQ ID NO:2 is also provided. In a specificembodiment, the polypeptide is one capable of associating with thecytoplasmic domain of Fas.

Yet another aspect of the invention is to provide an isolatedpolypeptide comprising the sequence of SEQ ID NO:2, an allelic orspecies variation thereof, or a fragment thereof, wherein the isolatedpolypeptide is a fusion protein. A FAF1-GAL4 activation domain fusionprotein is specifically provided. In one aspect, the fusion proteincomprises a tag, or a product of a second gene or fragment of thatsecond gene product. A fusion protein is provided wherein the tag isGST, an epitope tag or an enzyme or wherein the second gene is lacz.

A different aspect of the invention is the provision of antibodies thatspecifically bind a polypeptide comprising the sequence of SEQ ID NO:2,an allelic or species variation thereof, or a fragment thereof. Theseantibodies can be polyclonal, such as the rabbit antiserum providedherein, or monoclonal. A hybridoma capable of producing a monoclonalantibody to a FAF1 polypeptide is provided. Also provided is a kitcomprising any antibody preparation to the above-mentioned polypeptides.

In another aspect of the invention, a method is provided for isolating aFAF1 gene or fragment thereof, comprising screening a DNA library usinga FAF1 probe to identify a hybridizing clone and isolating said FAF1gene or gene fragment from said hybridizing clone. A FAF1 probe suitablefor use in this method is one which comprises the nucleotide sequence ofSEQ ID NO:1 or a fragment thereof. The method is useful to isolate ahuman FAF1 gene as well as FAF1 genes from other species.

Another aspect of the invention is to provide a method of modulating orblocking Fas activity comprising providing a Fas-interacting domainpolypeptide of FAF1 in a cell expressing Fas protein wherein saidFas-interacting domain polypeptide of FAF1 binds to said Fas protein toblock Fas activity. In one embodiment, the Fas-interacting domainpolypeptide of FAF1 is provided by introducing an expression vectorencoding a Fas-interacting domain polypeptide of FAF1 into the Fasexpressing cell. A method of activating FAF1-mediated apoptosis in acell is disclosed, the method comprising providing a constitutivelyactive FAF1 to the cell.

An important aspect of the invention is a method of screening for anagonist or an antagonist of FAF1, comprising contacting a cellexpressing both Fas and FAF1 with a test molecule, activating Fas, andanalyzing the cell for any effects on apoptosis, increased apoptosisindicative that the test molecule is an agonist and decreased or loss ofapoptosis indicative that the test molecule is an antagonist. The testmolecules can be peptides, oligonucleotides, lipids, toxins, hormones,small proteins, drugs and compounds from plant or animal sources andrecombinantly produced substances. In a specific embodiment, peptidelibraries are screened.

The cells are analyzed for effects on apoptosis such as cell membraneblebbing and/or DNA fragmentation. Fas is activated by binding to Fasligand or crosslinking with antibodies. Fas and/or FAF1 can be expressedas a fusion protein. In a particular embodiment, the cell expressesCD4/fas and HA epitope tagged FAF1 fusion proteins. An L cell expressingboth these fusion proteins is specifically provided in the invention.

The invention further provides a pharmaceutical composition useful inthe treatment of a disease resulting from dysregulated apoptosis,comprising a FAF1 polypeptide and a pharmaceutically acceptable carrier.Instead of the FAF1 polypeptide, the pharmaceutical composition cancomprise an expression vector capable of expressing the FAF1 polypeptidein an affected cell. The diseases contemplated for such treatmentinclude cancer, autoimmune disease and viral infections. For thesediseases, a constitutively active FAF1 polypeptide can be provided inthe pharmaceutical composition. In a different set of diseasescomprising myocardial infarction, stroke and reperfusion injury, arrestor blockage of apoptotic cell death is targeted. In the latter set ofdisease conditions, it may be therapeutically effective to express aFas-interacting domain polypeptide in the affected cell to block theinteraction between endogenous Fas and FAF1, thus blockingFAF1-potentiated apoptosis. Also provided are methods of alleviating apatient suffering from the aforementioned diseases by administering tothe patient, a therapeutically effective amount of the abovepharmaceutical compositions.

Finally, the invention provides several methods of isolating aFAF1-interacting protein. One method requires contacting a cell lysatesuspected of containing a FAF1 interacting protein with a FAF1polypeptide and isolating any protein bound to the FAF1 polypeptide, asa FAF1-interacting protein. A second method comprises labeling thecellular proteins, activating Fas expressed on the cell,immunoprecipitating FAF1 from the cell lysate and identifying a labeledprotein that coimmunoprecipitates with FAF1. In a third method, apeptide library is exposed to a FAF1 protein to allow one or morepeptides to bind to the FAF1 protein. Any bound peptide will be isolatedas a FAF1-interacting protein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1D shows surface expression of CD4/fas and CD4/fas786A detectedby fluorescent activated cytometry scanning (FACS) analysis.

FIG. 2 shows fragmentation of DNA from CD4/fas786A-23 cells (lanes 1-3)and from CD4/fas-16 cells (lanes 4-6) after anti-CD4 crosslinking. Lanes1 and 4 show DNA from control cells incubated with actinomycin D (0.5ng/ml) only. Lanes 2 and 5 show DNA from cells crosslinked with L3T4 Ratanti-CD4 (GK1.5) (1 μg/ml) alone. Lanes 3 and 6 show DNA from cellsincubated with both L3T4 (1 μg/ml) and anti-Rat IgG (0.5 μg/ml of Rabbitanti-Rat IgG, Zymed).

FIGS. 3A-3B shows the morphology of cells after anti-CD4 crosslinking.Cells were crosslinked as described as in FIG. 2 and photographs weretaken ten hours later. Top panel (FIG. 3A) shows CD4/fas-16 cells,bottom panel (FIG. 3A) shows CD4/fas786A-23 cells.

FIGS. 4A-4F shows the nucleotide sequence (SEQ ID NO:1) of the cDNAencoding FAF1.

FIGS. 5A-5D is an amino acid sequence (SEQ ID NO:2) deduced from thenucleotide sequence of the FAF1 cDNA.

FIG. 6A shows expression of FAF1 in Cos cells detected in whole celllysates by Western blot analysis using anti-HA epitope antibody (12CA5).The arrow indicates HA-tagged FAF1. In FIGS. 3A-6C, the lanes are asfollows: Cos cells were transfected with PSM plus PCGN8.1 (lane 1),PSMCD4/fas plus PCGN (lane 2), PSMCD4/fas plus PCGN8.1 (lane 3) orPSMCD4/fas786A plus PCGN8.1 (lane 4).

FIG. 6B shows a Western blot analysis blotting with 12CA5 antibody afterimmunoprecipitation of CD4.

FIG. 6C shows immunoprecipitated chimeric molecules of CD4/fas orCD4/fas786A detected by Western blot. The blot from FIG. 6B wasre-probed with anti-CD4 serum (a gift of Dr. D. R. Littman).

FIGS. 7A-7L show the morphological changes of cells expressing FAF1after CD4/fas antibody crosslinking. FIGS. 7A-7F show CD4/fas-16 cells.G-L show CD4/fas786A-23 cells. FIGS. 7A-7C and 7G-7I show cellstransiently expressing FAF1. D-F and J-L show cells mock transfectedwith vector alone. CD4/fas-16 and CD4/fas786A-23 cells wereco-transfected with PSV-β Gal and PCGN8.1 or PCGN alone (1:5 ratio) byDEAE Dextran method or Lipofectomin (BRL). Forty-eight to seventy-twohours later, transfectants were crosslinked by 1 μg/ml (FIGS. 7C, 7F,7I, and 7L) or 200 ng/ml (FIGS. 7B, 7E, 7H, and 7K) of L3T4 or a controlrat IgG (FIGS. 7A, 7D, 7G, and 7J) as described in FIG. 2. The cellswere fixed and photographed one hour later.

FIG. 8 shows the percentage of cells undergoing apoptosis 1 hour afterL3T4 treatment. The fixed cells, from the experiment described in FIGS.4A-4F, were assayed for β-galactosidase expression and blue cells werecounted by light microscopy. The percentage of apoptotic cells is thenumber of cells with cell membrane blebbing among one hundred blue cellscounted. The bars indicate means and standard deviations for fourindependent experiments.

DESCRIPTION OF THE PREFERRED EMBODIMENT

Fas antigen, a member of TNF/NGF (Tumor Necrosis Factor/Nerve GrowthFactor) receptor family, is a cell surface protein that mediatesapoptosis. Fas induces apoptosis when activated by Fas ligand (FasL)binding or anti-Fas antibody crosslinking (Nagata, S., Seminars inImmunol., 6:3-8 (1994)). Fas plays an important role in the developmentand function of the immune system (Nagata, supra, Lowin et al., Nature,370:650-652 (1994)). Malfunction of the Fas system causeslymphoproliferative disorders and accelerates autoimmune diseases,whereas exacerbation of Fas activity causes tissue destruction.

A point mutation in the cytoplasmic domain of Fas (a single base pairchange from T to A at base number 786), replacing isoleucine withasparagine, abolishes the apoptotic signal transducing property of Fas(Watanabe-Fukunaga et al., Nature, 356:314-317 (1992)). Mice homozygousfor this mutant allele (lpr^(cg) /lpr^(cg) mice) develop lymphadenopathyand an autoimmune disease that resembles human systemic lupuserythematosus (Watanabe-Fukunaga, supra). They produce large quantitiesof IgG and IgM including autoantibodies such as anti-DNA, and rheumatoidfactor (Cohen et al., Annu. Rev. Immunol., 9:243 (1991)) and developnephritis or arthritis. Patients have been described with phenotypessimilar to that of lpr mice (Sneller et al., J. Clin. Invest., 90:334(1992)) and patients with altered Fas were recently reported(Rieux-Laucat et al., Abstracts of the 12th European Immunology Meeting,Barcelona (European Federation of Immunological Societies) (June 1994)).

The present invention identifies a novel, Fas-associated factor 1,termed FAF1. FAF1, which was isolated using the two-hybrid screen inyeast (Durfee et al., Genes Dev., 7:555-569 (1993), specificallyinteracts with the cytoplasmic domain of wild type Fas but not thelpr^(cg) mutated Fas protein. This interaction occurs not only in yeastbut also in mammalian cells. When expressed in L cells, FAF1 potentiatedFas-induced apoptosis. A search of available DNA and protein sequencedata banks did not reveal any significant homology between FAF1 andother known proteins. Therefore, FAF1 is a novel protein that binds tothe wild type but not the inactive point mutant of Fas; it potentiatesFas-induced cell killing and is a signal transducing molecule in theregulation of apoptosis.

Blockage or activation of FAF1 function in apoptosis can be used in thetreatment of diseases including cancer, immune disorders such asautoimmune diseases, infectious diseases, and myocardial infarction andneuronal infarction in cardiovascular diseases.

Characterization of Apoptosis

The characteristics of cell death by apoptosis include cytoplasmic andnuclear condensation, loss of membrane integrity and extensivefragmentation of chromosomal DNA. The degraded DNA from apoptotic cellsforms a characteristic ladder when analyzed by gel electrophoresis(Vaux, D., Proc. Natl. Acad. Sci., 90:786-789 (1993)).

Yeast Two Hybrid Screening

The yeast two-hybrid system (Durfee et al., Genes Dev., 7:555-569(1993)) can be utilized to detect proteins capable of interacting with aknown protein. Briefly, the method is as follows. Plasmids areconstructed to encode two hybrid proteins which are coexpressed inSaccharomyces cerevisiae. One hybrid consists of the DNA-binding domainof the yeast transcriptional activator protein GAL4, fused to the knownprotein; the other hybrid consists of the GAL4 activation domain fusedto protein sequences encoded by a library of DNA fragments. Interactionbetween the known protein and a protein encoded by one of the libraryplasmids leads to transcriptional activation of a reporter genecontaining a binding site for GAL4. A suitable reporter gene is theSaccharomyces cerevisiae HIS3 gene and the E. coli lacZ gene (encodingβ-galactosidase (β-gal)). Yeast cells are tested for growth in medialacking histidine and for expression of β-gal activity which can beassayed by detecting blue colonies on a plate containing the substrate5-bromo-4-chloro-3-indolyl β-D-galactoside.

In the present invention, the hybrid construct encoding the knownprotein consists of the λ repressor dimerization domain/Fas cytoplasmicdomain chimera fused to the DNA-binding domain of GAL4. Using thistwo-hybrid screening system, cDNA clones encoding a protein thatinteracted with the Fas fusion molecule were isolated. A probe preparedfrom the longer cDNA clone was used to screen a murine thymus cDNAlibrary from which two full length cDNA clones encoding FAF1 wereisolated.

Definitions

As used herein, a "Fas-associated factor 1" is a protein which has anaffinity for Fas and binds or physically interacts with Fas.

A "FAF1 interacting molecule" or "FAF1 associating molecule" is amolecule which has an affinity for FAF1 and binds or physicallyinteracts with FAF1. If the interacting molecule is a protein, it isreferred to herein as "FAF1 interacting protein". The interaction can betransient, lasting only a fraction of a second or it can be stable so asto enable the detection of the complex of FAF1--FAF1 interactingmolecule. Preferably, this interaction persists for at least tenseconds, ideally several minutes. The term "FAF1 interacting molecule"does not imply any particular molecular size or other structural orcompositional feature other than that the molecule or compound inquestion is capable of binding or otherwise interacting with FAF1. Theinteracting molecule may be a substrate of FAF1, an enzyme that acts onFAF1, a protein that FAF1 is involved in localizing, an effectormolecule of FAF1 and/or Fas or a molecule that alters the conformationof FAF1 upon interaction. Interacting or associating molecules that canbe investigated by this invention include but are not restricted toagonists and antagonists of FAF1, cellular proteins encoded by oncogenesor proto-oncogenes, lipids, toxins, hormones, sugars, cofactors,peptides, proteins, enzyme substrates, drugs and compounds from plant oranimal sources.

A "Fas-interacting domain polypeptide or peptide of FAF1" is defined asa polypeptide or peptide having a sequence corresponding to a region inthe wild-type FAF1 protein, which physically interacts with thecytoplasmic region of Fas. The peptide will typically be in the range of10-30, preferably 25, amino acids.

An "isolated nucleic acid" is a nucleic acid, e.g., an RNA, DNA, or amixed polymer, which is substantially separated from other genome DNAsequences as well as proteins or complexes such as ribosomes andpolymerases, which naturally accompany a native sequence. The termembraces a nucleic acid sequence which has been removed from itsnaturally occurring environment, and includes recombinant or cloned DNAisolates and chemically synthesized analogues or analogues biologicallysynthesized by heterologous systems. A substantially pure moleculeincludes isolated forms of the molecule. An "isolated polypeptide" orprotein carries a similar meaning with the polypeptide or protein beingsubstantially separated from any cellular contaminants and componentsnaturally associated with the protein in vivo.

An "allelic variation" in the context of a nucleic acid or a gene is analternative form (allele) of a gene that exists in more than one form inthe population. At the polypeptide level, "allelic variants" generallydiffer from one another by only one, or at most, a few amino acidsubstitutions. A "species variation" of a nucleic acid or a polypeptideis one in which the variation is naturally occurring among differentspecies of an organism.

A "fragment" of a nucleic acid is a stretch of at least about 18nucleotides, more typically at least about 50 to 200 nucleotides butless than 2 kb. A polypeptide "fragment" or "segment" is a stretch ofamino acid residues of at least about 6 contiguous amino acids from aparticular sequence, more typically at least about 12 amino acids butcan be up to 20 amino acids.

The term "recombinant" or "recombinant DNA molecule" refers to a nucleicacid sequence which is not naturally occurring, or is made by theartificial combination of two otherwise separated segments of sequence.By "recombinantly produced" is meant artificial combination oftenaccomplished by either chemical synthesis means, or by the artificialmanipulation of isolated segments of nucleic acids, e.g., by geneticengineering techniques. Such is usually done to replace a codon with aredundant codon encoding the same or a conservative amino acid, whiletypically introducing or removing a sequence recognition site.Alternatively, it is performed to join together nucleic acid segments ofdesired functions to generate a single genetic entity comprising adesired combination of functions not found in the common natural forms.Restriction enzyme recognition sites are often the target of suchartificial manipulations, but other site specific targets, e.g.,promoters, DNA replication sites, regulation sequences, controlsequences, or other useful features may be incorporated by design."Recombinant DNA molecules" include cloning and expression vectors.

Two nucleic acids share sequence "identity" or "homology" if the twonucleic acids or designated segments thereof, when optimally alignedwith appropriate nucleotide insertions or deletions, are identical in atleast about 50% of the nucleotides. "Substantial homology" in thenucleic acid context means that the nucleic acids or their complementarystrands, when compared, are identical when optimally aligned withappropriate nucleotide insertions or deletions, in at least about 60% ofthe nucleotides, usually at least about 70%, more usually at least about80%, preferably at least about 90%, and more preferably at least about95 to 98% of the nucleotides. Alternatively, substantial homology existswhen the segments will hybridize under selective hybridizationconditions, to a strand, or its complement, using a sequence derivedfrom the FAF1 nucleic acids. Selectivity of hybridization exists whenhybridization occurs with a certain degree of specificity rather thanbeing random. Typically, selective hybridization will occur when thereis at least about 55% homology over a stretch of at least about 14nucleotides, preferably at least about 65%, more preferably at leastabout 75%, and most preferably at least about 90%. See, Kanehisa, Nuc.Acids Res., 12:203-213 (1984).

The technique of "polymerase chain reaction," or "PCR," as used hereingenerally refers to a procedure wherein minute amounts of a specificpiece of nucleic acid, RNA and/or DNA, are amplified as described inU.S. Pat. No. 4,683,195 issued Jul. 28, 1987. Generally, sequenceinformation from the ends of the region of interest or beyond needs tobe available, such that oligonucleotide primers can be designed; theseprimers will be identical or similar in sequence to opposite strands onthe template to be amplified. The 5' terminal nucleotides of the twoprimers may coincide with the ends of the amplified material. PCR can beused to amplify specific RNA sequences, specific DNA sequences fromtotal genomic DNA, and cDNA transcribed from total cellular RNA,bacteriophage or plasmid sequences, etc. See, generally, Mullis et al.,Cold Spring Harbor Symp. Quant. Biol., 51:263 (1987); Erlich, ed., PCRTechnology, (Stockton Press, NY, 1989). As used herein, PCR isconsidered to be one, but not the only, example of a nucleic acidpolymerase reaction method for amplifying a nucleic acid test sample,comprising the use of a known nucleic acid (DNA or RNA) as a primer.

"Operably Linked" means that a polynucleotide sequence it is placed intoa functional relationship with another polynucleotide sequence. Forexample, a promoter is operably linked to a coding sequence if it actsin cis to modulate the transcription of the linked sequence. Generally,"operably linked" means that the DNA sequences being linked arecontiguous and in the case of a secretory leader, contiguous and inreading phase. However, enhancers do not have to be contiguous. Linkingis accomplished by ligation at convenient restriction sites. If suchsites do not exist, then synthetic oligonucleotide adaptors or linkersare used in accord with conventional practice.

A "primer" or "oligonucleotide" can be a single-stranded polynucleotidethat may be chemically synthesized by known methods. Suitableoligonucleotides may be prepared by the phosphoramidite method describedby Beaucage and Carruthers, Tetr. Lett., 22:1859 (1981), or by thetriester method, according to Matteucci et al., J. Am. Chem. Soc.,103:3185 (1981), or by other methods such as by using commercialautomated oligonucleotide synthesizers such as Applied Bio Systemsoligonucleotide synthesizer, according to the specifications provided bythe manufacturer.

"Epitope" includes any protein determinant capable of specific bindingto an immunoglobulin or T-cell receptor. Epitopic determinants usuallyconsist of chemically active surface groupings of molecules such asamino acids or sugar side chains and usually have specific threedimensional structural characteristics, as well as specific chargecharacteristics.

As used herein, "constitutively active FAF1" means that FAF1 isfunctionally active independent of prior activation of Fas. Cellsexpressing the constitutively active FAF1 will exhibit induced apoptosiswithout prior activation of Fas by Fas ligand binding or Fascross-linking. "Constitutively active FAF1" shall be deemed to includefunctional derivatives thereof or homologs thereof of the wild-type FAF1protein. Derivatives can be produced by modifying any region in thewild-type protein such as by deleting negative regulatory regions or bymaking amino acid substitutions.

Generally, the nomenclature used hereafter and the laboratory proceduresin cell culture, molecular genetics, and nucleic acid chemistrydescribed below are those well known and commonly employed in the art.Standard techniques such as described in Sambrook et al., MolecularCloning, A Laboratory Manual, 2nd edition, Cold Spring HarborLaboratory, Cold Spring Harbor, New York (1989), are used forrecombinant nucleic acid methods, nucleic acid synthesis, cell culture,and transgene incorporation, e.g., electroporation, injection,lipofection. Generally enzymatic reactions, oligonucleotide synthesis,and purification steps are performed according to the specifications.The techniques and procedures are generally performed according toconventional methods in the art and various general references which areprovided throughout this document. The procedures therein are believedto be well known in the art and are provided for the convenience of thereader.

Specific Embodiments

Nucleic Acid and Polypeptide Compositions

The present invention provides an "isolated nucleic acid" encoding anovel, Fas-associated factor 1, defined herein as FAF1. The nucleotidesequence of the cDNA encoding full length FAF1, SEQ ID NO:1, is shown inFIGS. 4A-4F and the amino acid sequence, SEQ ID NO:2, is shown in FIGS.5A-5D.

Specifically provided by the invention is a nucleic acid comprising atleast 85% sequence identity, preferably 90%, most preferably 95% to 98%sequence identity with the nucleotide sequence of SEQ ID NO:1, an"allelic or species variation" thereof, or a fragment thereof. Thenucleic acid compositions of the present invention, whether RNA, CDNA,genomic DNA or a hybrid of the various combinations, can be isolatedfrom natural sources or may be synthesized in vitro.

The invention also provides certain recombinant DNA molecules comprisingthe nucleotide sequence of SEQ ID NO:1 or a fragment thereof, such asthe expression construct PCGN8.1 encoding FAF1 tagged at the N-terminuswith the HA epitope, and the FAF1/GAL4 transactivation domain fusionconstruct.

Compositions of the FAF1 polypeptide and derivatives thereof are alsoprovided. These compositions will be full length natural forms, thenatural forms including allelic and species variations of thepolypeptide encoded by SEQ ID NO:2, fragments of the natural forms,fusion proteins with those fragments and modified forms of each. Fusionproteins based on the FAF1 sequence include the HA epitope-tagged FAF1and the FAF1/GAL4 fusion protein used in the yeast two-hybrid systemdescribed in the experimental examples. FAF1 polypeptides will generallybe synthesized by expression from recombinant DNA molecules.Alternatively, FAF1 fragments and peptides can be synthesized bychemical methods such as described in Merrifield, B., Science, 232:342(1986); Kent, S. B. H., Ann. Rev. Biochem., 57:957 (1988); Offord, R.E., Semisynthetic Proteins, Wiley Publishing (1980); and Atherton etal., Solid Phase Peptide Synthesis, A Practical Approach, OxfordUniversity Press, Oxford (1989).

In a preferred embodiment, the FAF1 polypeptide or fragment thereof iscapable of associating with the cytoplasmic domain of Fas. In addition,pharmaceutical compositions are provided that include the FAF1polypeptide and its derivatives with a pharmaceutically acceptablecarrier.

Uses of FAF1 Nucleic Acids

The FAF1 nucleic acid and fragments thereof have various uses. In oneaspect of the invention, the mouse full length FAF1 nucleic acidaccording to the sequence of SEQ ID NO:1, or fragments thereof, are usedto prepare probes to screen DNA libraries to isolate FAF1 genes or genefragments encoded by other species, particularly human. The methods ofscreening DNA libraries are generally well known, see, e.g., Sambrook etal. (1989). If less than the full length FAF1 sequence is used, theprobes will be oligonucleotides or DNA fragments having at least about25 nucleotides, more usually at least about 100 nucleotides, and fewerthan about 2 knt (kilonucleotides), usually fewer than about 0.5 knt.Preferably, the probe should be free of vector sequences. The probes aretypically prepared labeled. Radiolabels such as ³² P are normally usedalthough non-radioactive labels are also suitable.

Conditions for hybridization can be varied. Initially, less stringentconditions can be used. However, if a high background of non-specifichybridization is observed, more stringent conditions will be employed.

The screening of mammalian cDNA or genomic DNA libraries, especiallyhuman DNA libraries, can be targeted although eukaryotes such as yeastand insects are also of interest for evolutionary comparisons. The DNAlibraries may be constructed in phage, bacteria or yeast. Clones thathybridize to the probe are identified such as by autoradiography ifradiolabeled probes are used. DNA is isolated from hybridizing clonesand analyzed for the presence of FAF1 gene sequences as verificationthat the hybridizing clone carries all or part of the FAF1 gene. The DNAsequence carried by the clone is compared with that of SEQ ID NO:1. TheFAF1 gene or fragment thereof will then be isolated from the vector byrestriction endonucleases. It may be necessary to isolate severaloverlapping DNA sequences from different hybridizing clones torecombinantly reproduce the full length gene in one contiguous DNAfragment.

Alternatively, with the availability of the mouse FAF1 sequence, FAF1genes from other species can be isolated by Polymerase Chain Reaction(PCR) by selecting appropriate pairs of primers based on the knownsequence and using genomic DNA or cDNA prepared from cells as thetemplate. Primers can be chemically synthesized and will be at least 10nucleotides in length, more usually 14 nucleotides, preferably 17nucleotides, but can be as long as 100 bp nucleotides. Pairs of primerscorresponding to the 5' and 3' ends of the gene or to the internalregions of the gene can be used. Several rounds of PCR may be requiredto prepare overlapping clones that can then be linked by recombinantmethods to produce the entire gene in one DNA fragment.

A gene that hybridizes with the probe and is determined to besubstantially homologous to the FAF1 gene in nucleotide sequence will beisolated. The homologous gene will be inserted into an appropriateexpression vector and introduced into a suitable host for expression toproduce the encoded polypeptide. The encoded polypeptide will then beassayed to determine if it associates with the appropriate species ofFas and functions like mouse FAF1, using the same procedure foranalyzing the interaction of FAF1 with Fas, described below in theExperimental Examples.

Other FAF1 structurally- or functionally-related family member proteinswill associate and interact with receptors similar to Fas and regulateapoptosis in other systems. The same library screening approach can alsobe used to identify other members of the family that share "substantialhomology" with FAF1.

The FAF1 probes can also be used to determine whether RNA encoding FAF1or a FAF1 homolog is present in a cell. This can be done by theprocedure of Northern Blotting. In situ hybridization can also beperformed on tissue sections of the organism to determine developmentalregulation and compare expression levels in various tissues. The probescan be labeled using any suitable labels or tags, e.g., radiolabel,biotin-avidin. The procedures of preparing probes, Southern blotting,Northern blotting and in situ hybridizations are well known in the art.See, for example, Sambrook et al., 1989.

The invention also provides a method of determining if the FAF1 genefrom a cell of interest is mutated. The cells can be from cultured celllines or from tissue isolated from an animal or human. For example,cells can be prepared from a human tumor biopsy. PCR can be used toamplify all or part of the FAF1 gene using selected primers and theamplified DNA fragment sequenced or analyzed for restriction enzymecleavage patterns. The nucleotide sequence or restriction analysis iscompared to the wild type sequence of FAF1 from the appropriate species.Therefore, the wild type sequence acts as a standard or positivecontrol.

Another aspect of the invention is the mapping of the functional domainsof FAF1. Functional domains of FAF1 include regions of FAF1 that:interact with Fas; interact with effector molecules; exhibit anenzymatic activity; or regulate the activity of FAF1 itself.Manipulating these domains of FAF1 can provide a means by which tocontrol Fas and FAF1 mediated apoptosis. For example, polypeptides orpeptides corresponding to the Fas-interacting domain of FAF1 can be usedto block Fas mediated apoptosis. Such polypeptides will generally be inthe range of 6 to 100 amino acids. Circumstances whereby blocking ofapoptotic cell death is desirable are described below under therapeuticuses.

There may be negative regulatory regions in FAF1 that control theactivation of FAF1 so that premature or unnecessary cell death does notoccur. These regulatory regions also serve as useful targets formutagenesis to affect the Fas apoptotic signaling pathway. For example,a constitutively active FAF1 could be produced by removing a negativeregulatory region. Such a modified FAF1 can be selectively expressed intargeted population of cells to affect cell death. This approach can beused to target elimination of cells that exhibit uncontrolledproliferation such as cancerous cells and autoreactive cells.

The method of mapping functional domains can be accomplished bygenerating a series of FAF1 deletion mutants which can then be comparedto the wild-type FAF1 in biological function. The deletion mutants willbe produced recombinantly using the full length FAF1 nucleic acid asstarting material, and more conveniently, the HA-epitope tagged FAF1described in the Experimental Examples. Tagging the mutant FAF1molecules either using the HA epitope or some other tag is desirable tofacilitate detection of the mutant and to distinguish the transfectedfrom the endogenous FAF1.

Generally, the mutants will have overlapping deletions. Initially,mutants will be created that have deletions typically in the range of10-50 amino acids. For subsequent more precise mapping, the deletions ina particular region will be smaller, typically in the range of 5-10amino acids. Mutants having single amino acid changes will be useful todefine key residues that determine structure or that may be involved inphysical contact with Fas or a downstream interacting or effectormolecule of FAF1. Such mutants can be generated by known techniques suchas by site-directed mutagenesis or by PCR.

Each deletion mutant is then transfected into an appropriate host celland the affects of each deletion on the function of FAF1, assessed. Tofacilitate analysis of the transfected FAF1, the host cell preferablyexpresses little or no endogenous FAF1 or expresses a mutated endogenousFAF1 that is non-functional. Preferably, mammalian cell lines amenableto transfection will be used and these include Cos and L cells.

The mutant transfected FAF1 will be analyzed for various characteristicsthat indicate increased, reduction or complete loss of Fas-mediatedfunction. For example, mutant FAF1 will be analyzed for physicalassociation with Fas by its ability to coimmunoprecipitate with Fas or aFas fusion polypeptide. The extent to which mutant FAF1 can potentiateFas-mediated apoptosis will be quantitated. Such assays are described inmore detail under Experimental Examples below.

The invention provides FAF1 nucleic acid and fragments thereof toprepare expression constructs for FAF1 and FAF1 polypeptide fragments.The expression vectors will contain the necessary elements fortranscription and translation of the DNA fragments into polypeptide ifthese elements are not already present in the DNA fragments themselves.These necessary elements include a promoter 5' of the DNA insert to beexpressed, a transcription and translation initiation site, stop codons,poly-A signal sequence, splice signals. DNA sequences encoding theprotein will be operably linked to a promoter appropriate for expressionin a particular cell type. Usually a strong promoter will be employed toprovide for high level transcription and expression. Examples of strongpromoters include human cytomegalovirus promoter. An enhancer may benecessary to function in conjunction with the promoter. The expressionconstruct normally comprises one or more DNA sequences encoding FAF1under the transcriptional control of a native or other promoter. Usuallythe promoter will be a eukaryotic promoter for expression in a mammaliancell, where the mammalian cell may or may not result in the expressionof FAF1. The selection of an appropriate promoter will depend upon thehost, but promoters such as the human cytomegalovirus promoter, viralLTRs such as SFFV LTR, and prokaryotic promoters such as trp, lac andphage promoters, tRNA promoters and glycolytic enzyme promoters areknown. In some circumstances, an inducible promoter may be preferred.See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Guide, Vols.1-3 (1989), Cold Spring Harbor Press.

Plasmid, viral or YAC vectors are contemplated. Conveniently availableexpression vectors which include the replication system andtranscriptional and translational regulatory sequences together withpolylinker restriction sites for insertion of the protein encodingsequence, may be employed. Such expression vectors are commerciallyavailable such as from Stratagene, Inc. Examples of workablecombinations of cell lines and expression vectors are described inSambrook et al. (1989); see also, Metzger et al., Nature, 334:31-36(1988).

It may be desirable to produce the FAF1 protein or fragments thereof ina prokaryotic host, in which case a prokaryotic promoter is preferred.Examples of prokaryotic promoters are trp, lac, and lambda. See Sambrooket al. (1989) for other useful prokaryotic promoters. Usually a strongpromoter will be employed to provide for high level transcription andexpression.

The expression construct will often be contained in a vector capable ofstable extrachromosomal maintenance in an appropriate cellular host ormay be integrated into the host genome. The expression construct may bebordered by sequences which allow for insertion into a host, such astransposon sequences, lysogenic viral sequences, or the like. Normally,markers are provided with the expression construct which allow forselection of host cells containing the construct. The marker ispreferably on the same DNA molecule but can be on a different DNAmolecule that is cointroduced into the host cell. In prokaryotic cells,markers such as a resistance to a cytotoxic agent, complementation of anauxotrophic host to prototrophy, production of a detectable product,etc., serve the purpose.

The expression construct can be joined to a replication systemrecognized by the intended host cell. Various replication systemsinclude viral replication systems such as retroviruses, simian virus,bovine papilloma virus, or the like.

While the wild-type sequences of FAF1 will generally be employed, insome situations one or more mutations or minor modifications may beintroduced, such as deletions, substitutions or insertions resulting inchanges in the amino acid sequence, providing silent mutations ormodifying amino acid residues or amino or carboxyl terminal groups.Conservative amino acid substitutions can be introduced. These aminoacid changes can be made using techniques such as PCR or site-directedmutagenesis.

There will be circumstances where gene fusions between FAF1 and anotherprotein can be useful. The fusion proteins will be recombinantlyproduced. The recombinant nucleic acid sequences used to produce fusionproteins of the present invention will often be derived from natural orsynthetic sequences. The FAF1 nucleic acid or fragments thereof find usein the construction of vectors encoding fusion proteins.

Fusion proteins encoding FAF1 fused to the GAL4 DNA binding domain canbe used in the yeast two hybrid system to isolate any proteins otherthan Fas that interact with the FAF1 protein, in particular, proteinsthat act downstream of FAF1. The yeast two-hybrid system is describedabove and in the experimental examples. This method allows the isolationof the cloned genes for the interacting proteins and eventually theidentification of the interacting proteins. Knowledge of the interactingproteins in the Fas signaling pathway will allow the screening of drugsfor agonists and antagonists of Fas dependent apoptosis.

The nucleic acid constructs will be useful to introduce into cells,providing an efficient and economical means to produce commerciallyuseful quantities of the protein compositions. Transfected cellsproducing varying quantities of full length FAF1 will also be useful inevaluating the effect of overexpression of FAF1 on Fas function andapoptosis. Nucleic acid constructs expressing various lengths and mutantforms of FAF1 can be used to determine structure-function relationships.

The means of introduction of the expression construct into a host cellwill vary depending upon the particular vector and the target host.Introduction can be achieved by any convenient means, including fusion,conjugation, transfection, transduction, electroporation, injection, orthe like. See, e.g., Sambrook, et al. (1989), supra. Transient or stabletransfection procedures can be used. The FAF1 nucleic acid compositionsare introduced into the appropriate cellular host under conditions whichfavor expression of the polypeptide and isolation of the resultantexpressed polypeptide. This implies using an expression vectorcompatible with the host cell, the vector containing the necessaryelements described above for expression of the polypeptide. Thetransfected cells are then provided with the optimum nutrient, gas andtemperature conditions for optimal protein production. These conditionswill depend on the cell type.

The host cells will normally be immortalized cells, i.e., cells that canbe continuously passaged in culture. For the most part, these cells willbe convenient mammalian cell lines which are able to express a FAF1protein and, where desirable, process the polypeptide so as to providean appropriate mature polypeptide and transport the mature protein tothe appropriate cell compartment. By processing is intendedglycosylation, ubiquitination, disulfide bond formation, generalpost-translational modification, or the like.

A wide variety of both prokaryotic and eukaryotic hosts will be employedfor expression of the proteins and peptides. Useful hosts includebacteria, such as E. coli., yeast, filamentous fungi, insect cells suchas Sf9, mammalian cells, typically immortalized, e.g., various mousecell lines, monkey cell lines, Chinese hamster ovary cell lines, humancell lines, derivatives of them, or the like. Since FAF1 appears to beubiquitous in expression by Northern analysis, it can be expressed inmost mammalian cell type. Cells that are amenable to transfection and invitro cell culture manipulation are preferred. In a specific embodiment,Cos and L cells are used. In a preferred embodiment, FAF1 and fragmentsthereof are expressed in tumor cells and cells of lymphoid, thymus orheart smooth muscle and epithelial cells of murine or human origin,depending on the FAF1 species used. In some cases, the cells will bederived from a neoplastic host cell or wild-type cells will betransformed with oncogenes, tumor causing viruses or the like. Cellscarrying the FAF1 nucleic acid compositions are covered by thisinvention.

Uses of FAF1 Transfected Cells

Cells transformed with the nucleic acid compositions can be used tocreate transgenic mice. Such transgenic mice are useful e.g. to studythe effect of overexpression of FAF1 on growth and development of theanimal. The procedure for producing transgenic mice is known in the artand are described in detail in Hogan et al., Manipulating the MouseEmbryo, Cold Spring Harbor laboratory, Cold Spring Harbor, N.Y. (1986).Mice having homozygous deletion of the FAF1 gene (knockout mice) canalso be generated by homologous recombination using gene targetingtechniques (see, e.g., Capecchi, Science, 244:1288-1292 (1989). Suchknockout mice are of interest to study the affect of FAF1 deficiency ondevelopment and cell differentiation.

Another aspect of the invention relates to methods of screening foragonists and antagonists of FAF1 which can affect apoptosis mediated bythe Fas pathway. The agonists and antagonists are preferably smallmolecules defined as being in the range of MW 5-20 kD and can cross theplasma membrane or be taken up by the cell. Test molecules will includeoligonucleotides, lipids, toxins, hormones, sugars, cofactors, peptides,small proteins, drugs and compounds from plant or animal sources andrecombinantly produced substances. FAF1 transfected cells (FAF1transfectants) are useful for such screening although cells naturallyexpressing Fas and FAF1 can also be used.

The screening method involves contacting a cell expressing both Fas andFAF1 with a test molecule, activating Fas, and analyzing the cell forany effects on apoptosis, increased apoptosis indicative that the testmolecule is an agonist and decreased or loss of apoptosis indicativethat the test molecule is an antagonist. Fas can be activated byproviding Fas ligand to the cell culture media or solution of the cellsample, to bind the Fas receptor on the cell surface. Alternatively, Fascan be activated by crosslinking with antibodies to the extracellulardomain of Fas. If Fas is expressed as a fusion protein, an antibodyspecific to the extracellular domain portion of the fusion protein willbe used to crosslink Fas. For example, an anti-CD4 antibody can be usedto crosslink a CD4/Fas fusion protein. FAF1 can be naturally orrecombinantly produced in the wild type form or expressed as a fusionprotein. Appropriate positive and negative control cells will berequired for comparison.

In one embodiment, L cells expressing a Fas fusion protein, such as theCD4/fas-16 cells described in the Experimental Examples, are furthertransfected with FAF1 or mock transfected with vector as described. Themock transfected CD4/fas-16 serve as negative control for the functionalassays. Transfectants expressing both Fas and FAF1 (test cells) and mocktransfectants expressing Fas only (negative control cells), arecontacted with a solution sample containing one or more test compounds.A sample of double transfectants will be treated under the sameconditions but in the absence of the test molecule. The cells can beexposed to the test compound added to the culture media. The testcompounds can initially be screened in pools comprising about 2-10different compounds or molecule types per sample solution.

Cells treated in the absence or presence of the test molecule will betested for eg. for disruption of Fas-FAF1 interaction or for any effecton apoptosis with or without stimulation of Fas. In one embodiment, thecells are crosslinked with anti-CD4 antibody (e.g., L3T4) and thenassayed for any effects on FAF1 activity essentially as described in theExperimental Examples under the subsection Association of FAF1 with Fas.The cells are analyzed for morphological changes such as cell membraneblebbing and other characteristics of apoptosis. Cellular DNA can beanalyzed for fragmentation by gel electrophoresis. Based on thesecriteria, the percentage of apoptotic cells in the test cells ascompared to similar cells treated in the absence of the test molecule orthe mock transfected cells can be determined. The test molecule isconsidered an agonist if the test cells contacted with the test moleculeshow increased frequency of apoptosis over the negative control. Incontrast, loss of or reduction in apoptosis in the test cells isindicative that the test molecule is an antagonist.

In a specific embodiment, peptide libraries expressing peptides of about8-15 amino acids, preferably 10-12 amino acids in length (10-12 kD), arescreened for agonistic or antagonistic effects on FAF1 activity. Thegeneration and screening of synthetic peptide combinatorial libraries isdescribed, e.g., in Houghten et al., Nature, 354:84-86 (1991). Librariesof peptide ligand can also be screened using the bacteriophage surfacedisplay technology as described by Djojonegoro et al., Biotechnology,12:169-172 (1994).

Isolation of FAF1 Polypeptide

The FAF1 polypeptide can be isolated from a normally expressing cell ora transfected cell by immunoprecipitation or affinity chromatography ofcell lysates using FAF1-specific antibody. The antibody can be insolution or affixed on a solid substrate. It may be more efficient toisolate the protein from transfected cells that may produce largerquantities of the protein due to particular characteristics of theexpression construct, such as a strong promoter. Instead of or inaddition to immunological methods, the peptide will generally beisolated by techniques employing FPLC, HPLC, electrophoresis, gradientcentrifugation and other methods routinely used in protein purificationto provide a substantially pure product, i.e., particularly free ofcellular contaminants. For protein purification methods, see, e.g.,Jacoby, Methods in Enzymology, Vol. 104 (1984), Academic Press, NewYork; Scopes, Protein Purification: Principles and Practice, (2nd Ed.)(1987) Springer-Verlag, New York; Deutscher (ed.), Guide to ProteinPurification, Methods in Enzymology, Vol. 182 (1990).

Uses of FAF1 Polypeptide Compositions

The FAF1 polypeptide compositions find several uses.

The polypeptide compositions of FAF1 are useful for raising antibodies,both polyclonal and monoclonal. Such antibodies are powerful tools thatcan be employed in various assays and diagnostic situations particularlywhere immunoprecipitation, immunoblotting and affinity purificationprocedures are necessary. Thus, one aspect of the invention is toprovide antibodies that specifically binds to the FAF1 polypeptidecomprising the sequence of SEQ ID NO:2, an allelic or species variationthereof, or a fragment thereof. Antibodies capable of specificallyblocking the binding of FAF1 to Fas are also desirable reagents. Theinvention provides a rabbit antiserum produced using the entire FAF1 asimmunogen. This rabbit anti-FAF1 antiserum is useful in bothimmunoprecipitating FAF1 and detecting FAF1 on Western blots. Theinvention also provides hybridoma lines that produce monoclonalantibodies to the FAF1 polypeptide.

These antibodies find use in isolating the FAF1 protein and anystructurally related proteins expressing an epitope recognized by theanti-FAF1 antibody. Isolation of FAF1 and structurally related proteinscan be accomplished by simply immunoprecipitating the proteins fromlysates of normally expressing or transfected cells. Alternatively,affinity purification of, e.g., cell lysates can be performed using theFAF1 antibody fixed on a solid matrix such as a column of beads or afilter paper.

FAF1 antibodies are also useful to study the interaction of FAF1 withFas in vivo, in normal and growth dysregulated or cancerous cells. Thesame protocol described in the experimental examples for studying theinteraction of FAF1 with Fas in intact cells can be followed.

Thirdly, the FAF1 specific antibodies find use in isolating any FAF1associating protein that co-immunoprecipitates with FAF1. FAF1associating proteins are useful to study the downstream effectors of Fasand the regulation of Fas and FAF1 function.

In a different aspect, FAF1 specific antibodies can serve as diagnosticreagents to detect deficiencies in FAF1 such as expression levels incells from patients suffering from certain autoimmune diseases or tumorcells from cancer patients.

Polyclonal and/or monoclonal antibodies with specificity to FAF1 can beprepared by in vitro or in vivo techniques following standard proceduresas described in, e.g., Harlow, et al., Antibodies: A Laboratory Manual(1988), Cold Spring Harbor Press, New York. Antibodies are produced byimmunizing an appropriate vertebrate host, e.g., rabbit or rodents, withthe entire FAF1 protein or peptides derived thereof, or in conjunctionwith an adjuvant. Usually two or more immunizations will be involved,and the blood or spleen will be harvested a few days after the lastinjection.

For immunization, preferably peptides corresponding to regions of theprotein comprising hydrophilic residues or residues exposed to theaqueous phase are selected. Immunogens comprising peptides correspondingto the region in FAF1 that interacts with Fas are desirable. Syntheticpeptide fragments may be prepared in a peptide synthesizer and coupledto a carrier molecule (e.g., keyhole limpet hemocyanin) and theconjugate injected into rabbits at selected times over several months.

For production of polyclonal antibodies, an appropriate host animal isselected, typically a rabbit or a mouse. The substantially purifiedantigen is presented to the immune system in a fashion determined bymethods appropriate for the animal and other parameters well known toimmunologists. Typical sites for injection are in the footpads,intramuscularly, intraperitoneally, or intradermally. Of course, anotherspecies will sometimes be substituted for a mouse or rabbit, includinggoats, sheep, cows, guinea pigs, and rats.

The rabbit or mouse sera is tested for immunoreactivity to the FAF1protein or peptide immunogen by an immunoassay, typically with preimmunesera as one of the negative controls. The immunoassay can be aradioimmunoassay, an enzyme-linked assay (ELISA), a fluorescent assay,or any of many other choices, most of which are functionally equivalentbut may exhibit advantages under specific conditions.

The polyclonal antibodies can be provided commercially in the form ofantisera or in purified form. From the polyclonal antisera, theimmunoglobulins may be precipitated, isolated and purified, such as byaffinity purification. Preferably, the purified form is substantiallyfree of non-specific antibodies and cellular contaminants.

Monoclonal antibodies with affinities of 10⁸ M⁻¹ preferably 10⁹ to 10¹⁰,or stronger will typically be made by standard procedures as described,e.g., in Harlow et al., Antibodies: A Laboratory Manual, CSH Laboratory(1988); or Goding, Monoclonal Antibodies: Principles and Practice (2ded) (1986), Academic Press, New York. Normally, mice are used to producemonoclonal antibodies although rats, guinea pigs and other animals canalso be used. After the appropriate period of time from the immunizationschedule, the spleens of such animals are excised and individual spleencells fused, typically, to immortalized myeloma cells under appropriateselection conditions. Thereafter the cells are clonally separated andthe supernatants of each clone are tested for their production of anappropriate antibody specific for the desired region of the antigen.

Other suitable techniques involve in vitro exposure of lymphocytes tothe antigenic polypeptides or alternatively to selection of libraries ofantibodies in phage or similar vectors. See, Huse et al., "Generation ofa Large Combinatorial Library of the Immunoglobulin Repertoire in PhaseLambda," Science, 246:1275-1281 (1989).

The polypeptides and antibodies of the present invention may be usedwith or without modification. Frequently, the polypeptides andantibodies will be labeled by conjugating, either covalently ornon-covalently, a substance which provides for a detectable signal.

A wide variety of labels and conjugation techniques are-known and arereported extensively in both the scientific and patent literature.Suitable labels include radionuclides, enzymes, substrates, cofactors,inhibitors, fluorescent moieties, chemiluminescent moieties, magneticparticles, and the like. Patents, teaching the use of such labelsinclude U.S. Pat. Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345;4,277,437; 4,275,149; and 4,366,241. Also, recombinant immunoglobulinsmay be produced, see Cabilly, U.S. Pat. No. 4,816,567.

The FAF1 antibodies of the invention can also be provided in a kit, withor without a FAF1 polypeptide, for use in any of the variousapplications described above. The contents of the kit will varydepending on the intended application of the antibody and may includeother reagents and instructions for the use of the antibody preparationand the reagents. At least one aliquot of the antibody will be provided.Different FAF1 antibodies with specificities for different regions ofthe protein can be provided. Different antibodies may be desirable forverification of an assay result. The aliquots can be contained in anysuitable container such as a vial or a tube. The polyclonal antibody canbe in the form of antisera or affinity purified. Monoclonal antibodiescan be provided in the form of ascites, culture media or a buffer suchas phosphate buffered saline solution. The antibody preparation can beprovided in solution or in lyophilized form, and may even be immobilizedon a substrate such as a column matrix. The antibody preparation mayalso contain in it preservatives such as sodium azide or proteaseinhibitors such as EDTA. A carrier protein such as BSA or ovalbumin,usually between 0.5-5%, may be included for stability. The solution formof the antibody, especially the purified form, may contain up to 50%glycerol if the kit is to be stored frozen at -20° C. to -70° C. If theantibody is provided in lyophilized form, the kit can include areconstitution buffer to reconstitute the antibody.

If the antibody is to be used in Western blot analysis, reagents for usein the Western blot procedure can be included in the kit. A secondary,labeled antibody capable of binding to the FAF1 antibody allowingdetection of the bound FAF1 antibody, can be included. The labeledantibody may be conjugated to an enzyme such as alkaline phosphatase orhorse radish peroxidase.

A FAF1 polypeptide included in the antibody kit will be useful as acontrol such as for molecular weight indicator on the blot and antibodydetection. FAF1 polypeptides by themselves can also be provided inseparate kits or vials for the various uses described. It is possiblethat FAF1 may have lipase activity. If FAF1 is enzymatically active, itmay be provided in a kit for use as an enzyme.

The invention also provides methods of isolating a FAF1 associatingprotein. As used herein, the terms "FAF1 associating protein" and "FAF1interacting protein" have the same meaning. A first method comprisescontacting a cell lysate suspected of containing a FAF1 associatingprotein with a FAF1 polypeptide and isolating any protein bound to saidFAF1 polypeptide as a FAF1 associating protein. The FAF1 polypeptide canbe isolated from cells that normally produce FAF1 at high levels or fromFAF1 transfected cells that overexpress FAF1. For this purpose, FAF1encompasses full length FAF1, fragments of FAF1 or FAF1 fusion proteins.A convenient source of FAF1 are the cells transfected with HAepitope-tagged FAF1 of the present invention.

In one embodiment of the first method, a FAF1 polypeptide is immobilizedon a solid matrix. The solid matrix can comprise various materials suchas is commonly used in column chromatography, including sepharose,sephadex, agarose, polystyrene and latex beads. The solid matrix canalso be filter paper or membrane, such as nitrocellulose andpolyvinylidene fluoride (PVDF) membrane. The FAF1 polypeptide can becoupled to the solid matrix directly or indirectly. Direct methodsinclude covalent coupling to sepharose beads using cyanogen bromide.Indirect coupling can take advantage of an FAF1 antibody or some othermoiety suitable for linking the two components such as biotin-avidinbinding pairs. FAF1 or FAF1 fusion proteins may be more convenientlyimmobilized if the fusion protein can bind a ligand provided on thematrix.

Lysates are prepared from FAF1 expressing cells. Cell lysates will becontacted with the immobilized FAF1 polypeptide such as by running thelysates over a FAF1 affinity column to allow any FAF1 interactingprotein to bind the immobilized FAF1 polypeptide under optimumconditions. Preferably the binding reaction is carried out between 4° C.and normal physiological temperature. Buffer conditions can be modifiedto favor capture of this binding. For example, the Ph and saltconditions can be varied. The matrix is then washed with buffer toremove any unbound or nonspecifically bound cellular components. Anyprotein that bound to the FAF1 polypeptide will be isolated by elutionoff the solid matrix such as by using a salt gradient or using solubleFAF1 polypeptide and fragments thereof to compete for binding.

In a second method, FAF1 expressing cells, preferably transfectants thatoverexpress FAF1, are biosynthetically labeled in order to label theproteins. For this method, the cells to be transfected with FAF1 or FAF1fragments are preferably cells that naturally express FAF1 so as tofavor the likelihood of the cells also expressing a FAF1 associatingprotein. It is likely that the Fas pathway has to first be activated toinduce a FAF1 interacting protein to associate with FAF1. Therefore, asample of cells treated to activate Fas will be tested in parallel. Asmentioned earlier, Fas can be activated by binding to Fas ligand (FasL)or by anti-Fas antibody cross-linking. If CD4/fas fusion proteinexpressing transfectants (such as CD4/fas-16 described below) are used,Fas can be cross-linked using anti-CD4 antibodies (e.g., L3T4). Thecells are lysed under different detergent conditions and FAF1 isimmunoprecipitated from the labeled cell lysate using anti-FAF1 antibodyor antiserum. Labeled proteins that co-immunoprecipitate with FAF1 areidentified by SDS-PAGE followed by autoradiography.

A third method for isolating FAF1 associating proteins involvesscreening a phage or bacterial peptide library for peptides capable ofbinding to FAF1 and isolating any bound peptide by affinitypurification. Fodor et al. in U.S. Pat. No. 5,143,854 describe methodsof preparing arrays of peptides on a solid matrix, screening of thepeptides and automated detection of peptides bound to ligand. The FAF1polypeptide can be provided in substantially pure form, immobilizeddirectly or indirectly onto a solid matrix. FAF1 can also be provided asan immunocomplex containing FAF1 bound to an antibody that specificallyrecognizes a tag on FAF1. A HA epitope tag and an anti-HA epitopeantibody combination is a suitable example. The tag antibody can becovalently bound to a solid matrix such as protein A-sepharose beads ordirectly conjugated to plain sepharose beads. Phage or bacterial peptidelibraries are exposed to the FAF1 protein to allow one or more peptidesto bind to the protein. Peptides which bind FAF1 are consideredassociated proteins and can be isolated by affinity purification.

Yet another approach to identifying FAF1 associating proteins is byusing the yeast two-hybrid system and FAF1 fusion proteins as describedearlier. Peptide expression libraries such as by phage displaymethodology, can be screened for ligand binding to the FAF1 polypeptideor fragments thereof or fusion proteins thereof.

FAF1 or FAF1 fusion proteins also find use to detect FAF1 interactingproteins by Western blotting, using the FAF1 protein in solution to bindcell lysate proteins immobilized on the blot and detecting the boundFAF1. In this circumstance, FAF1 protein can be labeled directly orindirectly. Indirect labeling will include, e.g., a labeled antibodybinding to FAF1.

FAF1 can be fused, e.g., with glutathione-s-transferase (GST), toproduce GST-FAF1 fusion proteins. Expression vectors carrying GSTsequence and specifically constructed to facilitate recombinantlyproducing GST fusion proteins are commercially available from, e.g.,Pharmacia. The fusion proteins may also comprise FAF1 or fragmentsthereof fused to the product or polypeptide encoded by a second gene. Aproduct encoded by only a portion or a fragment of the second geneinstead of the entire gene, may be sufficient. For example, FAF1 or afragment thereof may be fused to a second gene such as the E. coli lacZgene that will allow detection of expression. Other convenient fusionproteins will comprise FAF1 or fragments thereof linked to a tag.

In constructing fusion proteins, it will be understood that the amino orcarboxy terminus of the FAF1 protein, or wherever the fusion junctionis, may be modified to facilitate cloning or for other reasons, e.g., toallow cleavage of the fusion protein and release of the separateportions.

The tag can be a label or some means that allows identification of thefusion protein. The tag is introduced into a site in the polypeptidethat will not interfere with the folding and the function of theprotein, generally at the N- or the C-terminus. The tag can be anepitope tag recognizable by an antibody, a member of a binding pair, anenzyme or any other suitable entity. The tag can be a cleavable sequencesuch as the phosphatidylinositol-glycan (PIG) signal sequence present inproteins such as alkaline phosphatase, DAF and acetylcholinesterase. ThePIG sequence is cleavable by the enzyme phosphatidylinositolphospholipase C (PI-PLC) (Ferguson, Ann. Rev. Biochem., 57:285-320(1988)). The influenza virus hemagglutinin (HA) and the myc epitopes areparticularly useful tags. Examples of binding pairs are ligand-receptor,antigen-antibody and small molecules like avidin-biotin. Enzyme tagsinclude horse radish peroxidase, alkaline phosphatase andβ-galactosidase which can act on a substrate to produce a color signal.For example, the protein can be fused to an epitope tag recognizable byan available antibody. The antibody to the tag is useful. e.g., toimmobilize the FAF1 protein on an affinity column or to detect theprotein such as when the fusion protein is used in Western blotting.

The invention also provides a method to block or modulate FAF1's abilityto potentiate Fas-mediated apoptosis in vitro and in vivo. The methodinvolves using peptides corresponding to the Fas-interacting domain ofFAF1 to compete with endogenous wild-type FAF1 for associating with Fas.The Fas-interacting domain or region of FAF1 is determined as describedabove. By competing for binding to Fas, the Fas-interacting domainpeptide will block or reduce the ability of wild-type FAF1 to interactwith downstream effector molecules. This, in turn, will affect thecellular signalling events downstream of Fas.

The method of blocking Fas activity comprises providing aFas-interacting domain peptide of FAF1 in a cell expressing Fas and FAF1wherein the Fas-interacting peptide will bind to the Fas protein toblock Fas activity. The Fas-interacting domain peptide itself can bedirectly introduced into the cell under study where arrest or modulationof Fas function is desired. Methods of introducing the Fas-interactingdomain peptide into the cell include microinjection of the isolatedpeptide (expressed in other cells) or the use of appropriate drugdelivery vehicles such as liposomes to deliver the polypeptide.

Alternatively, the Fas-interacting domain peptide can be provided byintroducing an expression construct encoding the Fas-interacting domainpeptide into the desired cell wherein the Fas-interacting domain peptidewill be expressed in an amount effective to interfere with Fas activity.Expression constructs can be targeted to a particular cells by usingnucleic acid delivery vehicles that contain targeting moieties on thesurface of the vehicle. Examples of such vehicles include liposomes orrecombinant viruses expressing receptors for cell surface markers. Insome circumstances, complete blockage of Fas activity may require highlevel expression i.e. overexpression of the Fas-interacting domainpeptide for effective competition. In that circumstance, the expressionconstruct can be designed to contain the necessary elements such asstrong promoters, inducible promoters and enhancers to achieve highlevel expression of the Fas-interacting domain peptide. TheFas-interacting domain peptide expressed intracellularly will becontacted with and bind to the Fas protein.

Therapeutic Uses

An important embodiment of the invention is the treatment of diseases inwhich apoptosis is dysregulated. The treatments will have the potentialto change the natural progression of some of these diseases. Suchtherapeutic treatments will involve either the induction or theinhibition of apoptosis through FAF1, depending on the particulardisease condition. The invention provides therapeutic formulations andmethods of using these formulations to treat diseases.

The following lists examples of diseases where cells fail to undergoapoptotic cell death. In such diseases, it would be desirable to induceselective apoptosis in the affected cells so as to remove autoreactivecells, cancerous cells or viral infected cells that cause the disease.The diseases include: 1) Cancers, e.g., follicular lymphomas, carcinomaswith p53 mutations, hormone-dependent tumors such as breast cancer,prostate cancer and ovarian cancer; 2) Autoimmune disorders, e.g.,include systemic lupus erythematosus (SLE) and immune-mediatedglomerulonephritis; and 3) Viral infections such as caused byherpesviruses, poxviruses and adenoviruses. Such viral infectionsinclude fulminant hepatitis.

Several approaches for inducing or enhancing apoptosis are contemplatedin the present invention. One approach is to bypass Fas by providing aconstitutively active FAF1 that is functional in activating downstreamevents leading to apoptosis, independent of Fas activation by Fas ligandbinding or Fas antibody cross-linking. The generation of aconstitutively active FAF1 has been described above. The lpr^(cg)/lpr^(cg) mouse and human patients that manifest the lpr phenotype canbe used here as a model. Due to a mutation in Fas, the lpr^(cg)/lpr^(cg) mice develop lymphadenopathy and autoimmune disease andproduce large quantities of autoantibodies. The autoreactive B cells orcertain subsets of these B cells can be targeted for apoptotic celldeath by introducing a constitutively active FAF1 into these cells.

One method of providing a constitutively active FAF1 to a target cell isby gene therapy. A recombinant DNA molecule (expression vector) encodingthe constitutively active FAF1 is introduced into the cell whereby theactive FAF1 is expressed. The expression vector will be selectivelydelivered to the target cells only so as not to affect killing of allcells. Targeted delivery of DNA can be done for example by usingdelivery vehicles such as liposomes or viral vectors which havetargeting moieties on the surface. Liposome delivery vehicles withtargeting moieties are described in more detail below. Basically, thedelivery vehicle will contain on its surface, a targeting moiety thatrecognizes and binds a specific cell surface marker. In the example oftargeting delivery of the DNA to a specific clonal population of Blymphocytes, the membrane immunoglobulin expressed on the B lymphocyteserves as one convenient cell surface marker. The targeting moiety canbe an anti-idiotype antibody (the entire antibody or only the Fabregion) that recognizes the specific surface immunoglobulin expressed bythe B lymphocyte population. In this manner, the recombinant DNAexpression vector incorporated into the liposome can be specificallydelivered to the target cell population.

Recombinant viral vehicles can similarly be designed to express atargeting moiety on the viral capsid or envelope. It should be notedthat the targeting moiety can be a member of any binding paircombination and is not limited to that just antibody-antigen pairs. Theviral delivery vehicle will contain in its viral genome, the sequenceencoding the constitutively active FAF1. The recombinant viral DNA canbe designed to achieve targeted integration of the FAF1 sequence intothe genome of the target mammalian cell.

Other methods of introducing FAF1-encoding DNA into affected B cellsinclude local injection of naked DNA into lymph nodes or spleen.

The next set of diseases are associated with accelerated rates ofphysiological cell death and characterized by cell loss. In thesediseases, it would be desirable to provide therapeutic intervention toblock or modulate the exacerbated Fas-mediated apoptosis. The diseaseconditions include ischemic injury such as caused by myocardialinfarction (smooth muscle and epithelial cell death), stroke inducedneuron death and reperfusion injury; AIDS; and liver disease caused byviral infection, such as fulminant hepatitis.

Methods to block the activation of FAF1 through Fas include the use of(i) antagonists that are small molecules, (ii) peptide inhibitors and(iii) antisense inhibition. Again, liposomes can be used to targetdelivery of the small molecular and peptide inhibitors as well asantisense nucleic acids to specific cell populations.

Small molecule antagonists and peptide inhibitors have been discussedabove. Peptide inhibitors include peptides corresponding to regions ofFAF1 that interact with Fas or with effector molecules of FAF1 or thatexhibit an enzymatic activity. Peptide inhibitors are intended toinhibit interactions between Fas and FAF1, Fas and effector molecule orFas and substrate.

For a review of antisense therapy, see, e.g., Uhlmann et al., Chem.Reviews, 90:543-584 (1990). The antisense oligonucleotides will becomplementary to and hybridize specifically to the FAF1 MRNA, thusinhibiting translation of the mRNA and in addition, resulting in theRNAse H cleavage of the bound mRNA. The antisense oligonucleotides canbe DNA or RNA although DNA is preferred because of greater stability.They can be chemically modified so as to improve stability in the body.The antisense oligonucleotide will be at least 15 nucleotides butgenerally 17 nucleotides (MW about 5,000) or longer. RNAoligonucleotides can be provided in the form of ribozymes designed tocleave the mRNAs to which they bind. The ribozymes will comprise RNA ormixed RNA-DNA oligonucleotide.

The disease conditions described such as exacerbated cell death due tomyocardial infarction or stroke can be treated by administering to thepatient, a therapeutic formulation comprising an inhibitor of FAF1activity in an amount effective to block the FAF1 activity in affectedcells in the patient. The inhibitor of FAF1 activity can be aFas-interacting domain peptide. The patient can be administered atherapeutically effective amount of a pharmaceutical compositioncomprising a Fas-interacting domain peptide, and a pharmaceuticallyacceptable carrier. The Fas-interacting domain peptide will bespecifically targeted to the affected cells, such as cardiac smoothmuscle cells and epithelial cells in myocardial infarction, hepatocytesin fulminant hepatitis, and T cells and macrophages in AIDS. Anotherpharmaceutical composition for use in the treatment method will comprisean expression vector suitable for introduction into and expression of atherapeutically effective amount of a Fas-interacting domain peptide inthese aforementioned cells.

Drug delivery vehicles such as liposomes can be used to deliver andprovide sustained release of the formulations in the body. The liposomescan have targeting moieties exposed on the surface such as antibodies,ligands or receptors to specific cell surface molecules. For example, itmay be desirable to limit the delivery of the formulation to only tumorcells. Such cells can be targeted to receive the therapeutic formulationby incorporating into the liposome carrier, a targeting moiety thatrecognizes and binds a specific tumor surface marker. Liposome drugdelivery is known in the art (see, e.g., Biochimica et Biophysica Acta,113:201-227 (1992)).

The quantities of reagents determined to be an effective amount fortreatment will depend upon many different factors, including means ofadministration, target site, physiological state of the patient, andother medicants administered. Thus, treatment dosages should be titratedto optimize safety and efficacy. Typically, dosages used in vitro mayprovide useful guidance in the amounts useful for in situ administrationof these reagents. Animal testing of effective doses for treatment ofparticular disorders will provide further predictive indication of humandosage. Various considerations are described, e.g., in Gilman et al.(eds), Goodman and Gilman's: The Pharmacological Bases of Therapeutics,8th Ed. (1990), Pergamon Press; and Remington's Pharmaceutical Sciences,17th Ed. (1990), Mack Publishing Co., Easton, Pa. Methods foradministration are discussed therein, e.g., for oral, intravenous,intraperitoneal, or intramuscular administration, transdermal diffusion,and others.

If the pharmaceutical composition is formulated for oral delivery andcontains a peptide or peptide-like compound as the active agent, thenthe formulation must include a means for protecting the agent from theproteolytic enzymes of the digestive system. Typically, the agent isencased in a liposome structure or chemically derivatized so that theenzymes are prevented from cleaving the amide bonds of the peptide,resulting in the agent's degradation.

The pharmaceutical compositions will be administered by intravenous,parenteral, intraperitoneal, intramuscular, oral, or localadministration, such as by aerosol or transdermally, for therapeutictreatment. The pharmaceutical compositions can be administered in avariety of unit dosage forms depending upon the method ofadministration. For example, unit dosage forms suitable for oraladministration include powder, tablets, pills, capsules and dragees.

The pharmaceutical compositions will often be administeredintravenously. Thus, this invention provides compositions forintravenous administration which comprise a solution of the polypeptidedissolved or suspended in an acceptable carrier, preferably an aqueouscarrier. Slow release formulations, or slow release delivery vehicleswill often be utilized for continuous administration. "Pharmaceuticallyacceptable carriers" will include water, saline, buffers, and othercompounds described, e.g., in the Merck Index, Merck & Co., Rahway, N.J.These compositions will sometimes be sterilized by conventional, wellknown sterilization techniques, or may be sterile filtered. Theresulting aqueous solutions may be packaged for use as is, orlyophilized, the lyophilized preparation being combined with a sterileaqueous solution prior to administration. The compositions may containpharmaceutically acceptable auxiliary substances as required toapproximate physiological conditions, such as pH adjusting and bufferingagents, tonicity adjusting agents, wetting agents and the like, forexample, sodium acetate, sodium lactate, sodium chloride, potassiumchloride, calcium chloride, sorbitan monolaurate, triethanolamineoleate, etc.

For solid compositions, conventional nontoxic solid carriers may be usedwhich include, for example, pharmaceutical grades of mannitol, lactose,starch, magnesium stearate, sodium saccharin, talcum, cellulose,glucose, sucrose, magnesium carbonate, and the like. For oraladministration, a pharmaceutically acceptable nontoxic composition isformed by incorporating any of the normally employed excipients, such asthose carriers previously listed, and generally 10-95% of activeingredient, preferably about 20% (see, Remington's, supra).

The compositions containing the compounds can be administered forprophylactic and/or therapeutic treatments. In therapeutic applications,compositions are administered to a patient already suffering from adisease, as described above, in an amount sufficient to cure or at leastalleviate the symptoms of the disease and its complications. An amountadequate to accomplish this is defined as "therapeutically effectivedose." Amounts effective for this use will depend on the severity of thedisease and the weight and general state of the patient.

In prophylactic applications, compositions containing the compounds ofthe invention are administered to a patient susceptible to or otherwiseat risk of a particular disease. Such an amount is defined to be a"prophylactically effective dose." In this use, the precise amountsagain depend on the patient's state of health and weight.

The following examples are offered by way of illustration and are notmeant to be construed as a limitation on the scope of the invention.

EXPERIMENTAL EXAMPLES

Materials and Methods

Antibodies--GK1.5, used as a primary crosslinking antibody, is amonoclonal antibody specific to murine CD4 (L3T4) (Caltag). PE-L3T4(Caltag) is GK1.5 conjugated with PE and was used for surface stainingof CD4/fas expression by Fluorescence Activated Cytometry Scanning(FACS) analysis (FACS IV, Becton-Dickinson). A rabbit Anti-Rat IgG wasused as secondary crosslinking antibody (Zymed). 12CA5 is a monoclonalantibody specific for the hemaagglutinin (HA) epitope of the influenzavirus (Wilson et al., Cell, 37:767-778 (1984)). A rabbit serum specificfor murine CD4 (a gift from Dr. D. Littman) was used for detection ofCD4/fas by Western blot.

DNA Constructs--A chimeric molecule of CD4 and fas was subcloned intovector PSM described in Brodsky et al., J. Immunol., 144:3078-3086(1990), which has an SV40 replication origin and an SV40 early promoter.PSMCD4/fas contains the chimera with a wild type cytoplasmic domain ofFas. PSMCD4/fas786A has a T to A point mutation at base pair 786 in thecytoplasmic domain of fas. Fusion molecules of the λ repressordimerization domain and Fas cytoplasmic domain were inserted in framewith GAL4 DNA-binding domain and HA epitope in the vector PAS-CHY(Durfee et al., Genes Dev., 7:555-569 (1993)). FAF1 tagged with an HAepitope at the N-terminus was subcloned into the PCGN vector (with a CMVpromoter) to make PCGN8.1.

Cells, Transfections and Immunoprecipitation--For co-immunoprecipitationexperiments, Cos cells were transiently transfected with PCGN8.1 aloneor PCGN8.1 plus PSMCD4/fas or PCGN8.1 plus PSMCD4/fas786A by DEAEDextran method (Gorman, DNA Cloning, A Practical Approach, IRL Press,Oxford, Vol. II, pp. 143-190 (1985)). Transfectants were lysed by lysisbuffer (20 mM Tris. PH7.5, 137 mM NaCl, 1% triton×100) two days later.The expression level of FAF1 was quantitated by Western blot analysiswith 12CA5 antibody. Cell lysates were used for immunoprecipitation ofCD4 by GK1.5. Immune-complexes were analyzed by 8% SDS-PAGE andtransferred to nitrocellulose paper. The paper was then incubated with12CA5 antibody and developed by an alkaline phosphatase method(Boehringer Mannheim). To quantitate the amount of CD4/fas orCD4/fas786A immunoprecipitated, the blot was stripped off, re-probedwith anti-CD4 serum and then developed by Enhanced Chemiluminescence(ECL) method (Amersham).

L cells were transfected with PSMCD4/fas or PSMCD4/fas786A plus a TKpromoter driven neo gene expression vector (Stratagene) by calciumprecipitation. The cells were then selected in medium with G418 (400μg/ml) for 10 days (Itoh et al., Cell, 66:233-243 (1991)). Individualclones were analyzed for CD4 surface expression by FACS as follows.Single clones (1×10⁶ cells) of untransfected L cells (L cells), L cellstransfected with PSM vector alone (PSM-1), L cells transfected withPSMCD4/fas (CD4/fas-16) or with PSMCD4/fas786A (CD4/fas786A-23) wereincubated separately with 1 μl of fluorescence (PE) conjugated anti-CD4antibody (PE-L3T4, Caltag) in 100 μl PBS (3% FCS) at 4° C. for 30minutes, washed twice with PBS (3% FCS), and analyzed by FACS (FACS IV,Becton-Dickinson). Multiple clones expressing either the wild type ormutant chimera were generated.

For FAF1 functional studies, PCGN8.1 or PCGN vector plus PSV-βGAL (5:1ratio) was transiently transfected into both CD4/fas and CD4/fas786Aexpressing cells by either DEAE Dextran or Lipofectmin (BRL).Transfected cells were identified by their expression of β galactosidase(stained blue cells with X gal) (Harlow et al., Antibodies. A LaboratoryManual, Cold Spring Harbor Laboratory, p. 401 (1988)).

Antibody crosslinking and assays for apoptosis--CD4/fas or CD4/fas786Aexpressing cells were incubated with appropriate amounts of GK1.5 at 4°C. for 20-30 minutes and rinsed with 37° C. pre-warmed DME medium (0%serum). Antinomycin D (final concentration of 0.5 μg/ml) were added tothe cells. The cells were then incubated at 37° C. for different times.

To detect DNA fragmentation, DNA was extracted from cells two hoursafter crosslinking by lysing the cells with 1% SDS. The cell lysate wasdigested with proteinase K at 55° C. overnight and DNA precipitated withisoamyl alcohol. The extracted DNA was analyzed with molecular weightmarkers by electrophoresis in 2% agarose gel and staining with ethidiumbromide.

For FAF1 functional assays, the cells were crosslinked at 48 to 72 hoursafter transfection. Then the cells were visualized under the microscopeand photographs were taken at different times to detect changes incellular morphology. To quantitate the percentage of apoptic cell deathat a given time, the crosslinked cells were fixed, stained with Xgal andcounted for the number of apoptotic cells (membrane blebbing) in 100blue cells.

Fas-Mediated Apoptosis

In order to understand the mechanism of signal transduction inFas-mediated apoptosis, it was first determined whether the cytoplasmicdomain is sufficient to initiate an apoptosis signal. A chimeric cDNA(CD4/fas) containing the cytoplasmic domain of murine Fas linked to theextracellular and transmembrane domains of murine CD4 (CD4/fas) wasmade. As control, the point mutation of Fas in lpr^(cg) mice (T786A) wasalso made as an analogous chimeric molecule (CD4/fas786A). The chimeraswere stably transfected into L cells and clones expressing equivalentlevels of wild type (CD4/fas) or mutant (CD4/fas786A) chimeric moleculeswere chosen for analysis (FIGS. 1A-1D). L cells expressing CD4/fas(CD4/fas-16 underwent apoptotic cell death when crosslinked bymonoclonal antibody against CD4 (L3T4, Caltag)) in the presence ofactinomycin D (Itoh et al., DNA Cloning, a Practical Approach, IRLPress, Oxford. Vol. II, pp. 143-190 (1991). DNA fragmentation,characteristic of apoptosis, was observed two hours after the antibodycrosslinking (FIG. 2). Cells were shrunk and detached from the bottom ofthe culture dish at 10 hours (FIGS. 3A-3B). However, L cells expressingthe mutant chimera (CD4/fas786A-23), under the same treatment, did notundergo apoptotic cell death. Multiple clones of each type were analyzedand gave the same results. It was concluded that the cytoplasmic domainof Fas can initiate an apoptotic signal.

The results also showed that dimerization of the Fas cytoplasmic domainis sufficient to generate an apoptotic signal. The antibody L3T4(GK1.5), used here to induce apoptosis through the Fas cytoplasmicdomain, is a bivalent Rat IgG-2b. As shown in FIG. 2, there was nosignificant change upon the addition of a secondary anti-Rat IgGantibody, again indicating that dimerization was sufficient for Fasactivation.

Screening for Fas-Interacting Protein

An improved version of the yeast two-hybrid system (Durfee et al., GenesDev., 7:555-569 (1993) originally devised by Fields and Song, Nature,340:245-246 (1989)) was used to screen for Fas interacting proteins. Inorder to simulate activated dimeric Fas, a fusion molecule of the λrepressor dimerization domain and the Fas cytoplasmic domain wasconstructed. The fusion molecule was then linked to the DNA bindingdomain of GAL4 for two-hybrid screening. As a control, a similarconstruct was made with the T786A mutation (lpr^(cg) mutation) in theFas cytoplasmic domain.

The nucleotide and amino acid sequence of murine and human Fas isprovided in Watanabe-Fukunaga et al., J. Immunol., 148:1274-1279 (1992)and Itoh et al., Cell, 66:233-243 (1991), respectively. The Fas cDNAsequence of lpr^(cg) mice is disclosed in Watanabe-Fukunaga et al.,Nature, 356:314-317 (1992). The amino acid sequence of the Fascytoplasmic domain from wild-type mouse, cg mouse and human can also befound in Watanabe-Fukunaga et al., Nature, supra. λ repressordimerization domain sequence is disclosed in Amaya et al., Development,118:477-487 (1993).

The plasmid CIXD containing the λ repressor dimerization domain wasobtained from Marc Kirshner. Plasmid CIXD was digested with PvuII andBal I to release a 430 bp insert containing the dimerization domain.This insert was subcloned into the blueskript plasmid pBS at the Sma Isite. The insert was then excised from pBS using the convenient linkersites, BamHI and EcoRI, present in the vector. This BamHI-EcoRI fragmentwas blunt-ended with Klenow.

The plasmid pAS1.FasCyt contains the GAL4 DNA binding domain fused tothe Fas cytoplasmic domain. The BamHI-EcoRI blunt-ended fragmentcontaining the λ repressor dimerization domain was inserted intopAS1.FasCyt at a blunt-ended NdeI site in between the GAL4 domain andthe Fas cytoplasmic domain, producing an in-frame fusion of GAL4 DNAbinding domain-λ repressor dimerization domain-Fas cytoplasmic domain inthe construct pAS1/RDD/CYT. This ligation creates 4 new amino acids,RSPL (SEQ ID NO:3), at the junction of GAL4 and λ dimerization domain,and 6 new amino acids, GCRNSI (SEQ ID NO:4), between the dimerizationdomain and the Fas cytoplasmic domain. As a control, a similarconstruction was made using Fas having the T786A mutation (lpr^(cg)mutation) in the cytoplasmic domain.

A GAL4 transactivation domain-tagged cDNA expression library wasprepared using a murine T cell line cDNA library (a gift from Dr. S.Elledge). The yeast strains and vectors used herein as well as cDNAlibraries from different cell lines, linked to the GAL-4 transactivationdomain, are also commercially available from, e.g., Clontech Labs Inc.,Palo Alto, Calif. The yeast reporter strain is cotransformed with theλ/Fas/GAL4 DNA binding domain fusion construct and the T cell cDNAlibrary/GAL4 transactivation domain fusions.

More than 1.1 million clones of the T cell line cDNA library werescreened for their ability to interact with the Fas fusion molecule inthe two-hybrid system. Four independent clones interacted specificallywith the wild type Fas constructs but not the mutant Fas constructs. Twoclones had 2.2 kb inserts and two other had 2 kb inserts. Sequenceanalysis showed that all 4 clones were derived from the same gene andfused to the activation domain of GAL4 in the same reading frame. Theinserts of the shorter 2 kb clones were missing approximately 150 bp atthe 5' end and 50 bp at the 3' end of the sequence of the longer clones.A murine thymus CDNA library (a gift of Dr. M. Davis) was screened witha DNA probe consisting of 0.7 kb of the most 5' end of the 2.2 kb cloneisolated by the two-hybrid screening in yeast. Two independent cDNAclones of approximately 2.6 kb were obtained from the murine thymus cDNAlibrary.

Characterization of the FAF1 Sequence

Sequence analysis indicated that these were full length cDNAs andcontained an open reading frame encoding a protein of 649 amino acids(FIGS. 4A-4F). The deduced molecular weight is 74 kD and the PI was 4.6.The translation start site contains a perfect "Kozak" consensus sequence(Kozak, M., J. Cell Biol., 108:229-241 (1989)). There are two regions,amino acids 280 to 310 and 490 to 590, that are highly negativelycharged and have a predicated a-helical secondary structure. There arethree potential myristoylation sites located at amino acid 50, 306 and310. There are also three N-glycosylation sites at amino acid 163, 209and 423. No significant sequence homology of these clones was found withany complete protein sequence in available sequence data banks. Thisnovel protein was named Fas-associated factor 1 (FAF1).

Association of FAF1 with Fas in Mammalian Cells

To show that the association can occur in mammalian cells, an influenzavirus hemagglutinin (HA) epitope tagged FAF1 was transientlyco-expressed with CD4/fas or CD4/fas786A in Cos cells. The full length2.2 kb FAF1 cDNA was tagged at the N-terminus with a sequence encoding a15 amino acid HA epitope and cloned into a CMV promoter-drivenexpression vector PCGN to produce the construct PCGN8.1. The PCGN vectoris described in Klippel et al., Mol. Cell. Biol., 13:5560-5566 (1993).Cos cells were transfected with PSM plus PCGN8.1 (FIGS. 6A-6C, lane 1),PSMCD4/fas plus PCGN (lane 2), PSMCD4/fas plus PCGN8.1 (lane 3) orPSMCD4/fas786A plus PCGN8.1 (lane 4). Two days after transfection, thecells were lysed (1% triton×100, 20 mM Tris, pH 7.5, 137 mM NaCl plusproteinase inhibitors) and 0.5% of the lysates were used to determinethe FAF1 expression level by Western blot analysis with 12CA5 (FIG. 6A).Four μl of L3T4 was added to the rest of the lysates and incubated at 4°C. for one hour. 50 μgl of anti-Rat IgG conjugated agarose beads (Sigma)were added and incubated for another hour. The beads were washed threetimes with PBS (1% NP40). The immune-complexes were electrophoresed on8% SDS-PAGE and transferred to nitrocellulose paper. The paper was firstincubated with 12CA5 and developed by an alkaline phosphatase method todetect FAF1 (FIG. 6B). The antibody was then stripped off and the paperreincubated with anti-CD4 antibody and developed by ECL to detectCD4/fas (FIG. 6C).

FAF1 was expressed equally in the Cos cells expressing either CD4/fas orCD4/fas786A (FIG. 7A). By immunoprecipitating with anti-CD4 antibody andblotting for the HA epitope, a much greater quantity of FAF1 wasco-immunoprecipitated with CD4/fas than with CD4/fas786A (FIG. 6B),although more CD4/fas786A was immunoprecipitated than CD4/fas (FIG. 6C).As shown in FIG. 6B, the molecular weight of FAF1 detected on SDS-PAGEis 75-80 kD which is heavier than the predicated 74 kD. This could beaccounted for by the post-translational modifications such asglycosylations of the molecule. Thus, FAF1 was able to specificallyassociate with the cytoplasmic domain of wild type Fas in Cos cells.

To determine the significance of the association between FAF1 and Fas,FAF1 was transiently expressed in CD4/fas-16- or CD4/fas786A-23-stablytransfected L-cells. CD4/fas-16 and CD4/fas786A-23 cells wereco-transfected with PSV-β Gal and PCGN8.1 or PCGN alone (1:5 ratio) byDEAE Dextran method (Gorman, DNA Cloning, a Practical Approach, IRLPress, Oxford. Vol. II, pp. 143-190 (1985)) or Lipofectomin (BRL).Forty-eight to seventy-two hours later, transfectants were crosslinkedby different amounts of L3T4 (GK1.5) antibody in the presence ofactinomycin D: 1 μg/ml (FIGS. 7C, 7F, 7I, and 7L) or 200 ng/ml (FIGS.7B, 7E, 7H, and 7K) of L3T4 or a control rat IgG (FIGS. 7B, 7D, 7G and7J) as described in FIG. 2. The cells were fixed and photographed onehour later.

In CD4/fas-16 cells, transient expression of FAF1 resulted in more rapidand extensive apoptosis than in mock transfected cells (FIG. 7A). Onehour after addition of 200 ng/ml of L3T4 crosslinking antibody,approximately 60% of CD4/Fas-16 cells expressing FAF1 had undergoneapoptotic cell death compared with 30% in the cells without FAF1overexpression (FIG. 8). Increasing the L3T4 concentration to 1 μg/ml,increased the apoptotic cell death to approximately 70% and 40%respectively (FIG. 8). There was no obvious apoptosis observed inCD4/fas786A-23 cells treated similarly (FIGS. 7A-7L and 8). Apoptosisinduced through Fas was thus increased from 30%-40% in the controls to60%-70% when FAF1 was expressed. Similar results were obtained in ahuman T cell leukemia line, Jurkat, where transient expression of FAF1potentiated apoptosis induced by anti-human Fas antibody. These dataindicate that FAF1 can potentiate apoptosis mediated by Fas and actsdownstream of Fas.

The results indicate that FAF1 is a molecule which acts downstream inthe Fas signal transduction pathway. FAF1 can interact selectively withthe wild type cytoplasmic domain of Fas. This specific interactionoccurred not only in yeast cells but also in mammalian cells. Thebinding is not the result of overexpression of FAF1 in Cos cells becausethe level of FAF1 expression was relatively low (approximately onetenth) compared to other proteins that were expressed with the sameantibody tag. When expressed transiently in L cells, FAF1 was able topotentiate apoptosis induced by Fas.

All publications and patent documents cited herein are herebyincorporated by reference.

    __________________________________________________________________________    #             SEQUENCE LISTING    - (1) GENERAL INFORMATION:    -    (iii) NUMBER OF SEQUENCES: 4    - (2) INFORMATION FOR SEQ ID NO:1:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 2558 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: cDNA    -     (ix) FEATURE:              (A) NAME/KEY: CDS              (B) LOCATION: 413..2359    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:    - TCGAGCTACG TCAGGGCTGG AGGGAGCCGG GCGCGCGCTG TTCGCAACCT GT - #CCTCCTCC      60    - CAGGCGGCGA CGGAAGGACC GGCCCGGCAT CGAGACCAGC CTCCCTCGCA AC - #CTGTCCTC     120    - CTCCCAGGCG GCGACGGAAG GACCGGCCCG GCATCGAGAC CAGCCTCCCC GT - #CCCGGCAG     180    - CTGCGGCGAG GGCTCCGCGG GGCGCAGGCG GGCTCAGGGC GGCTGAAGGT TA - #CCGAGTGC     240    - ATGAGCACCT AGTCTCCCGC GCTGCCCCGC CCGCCGGTCC GCCGGCCCCT CC - #CGCCGGCT     300    - CGCCCGCCAG CCCTTCGCCA CCCGGCGGCG GCCGCAGCTT CGGCCGCAGG AG - #GCGCCGTC     360    - TCGCTCCCAG GTGCGCGCTT CGTTCCCGGA GCCGCGGAGC TCGGCGGCCG CC - # ATG     415    #    Met    #      1    - GCG TCC AAC ATG GAT TTA CCG ATG ATC CTT GC - #G GAT TTT CAG GCA TGT     463    Ala Ser Asn Met Asp Leu Pro Met Ile Leu Al - #a Asp Phe Gln Ala Cys    #              15    - ACT GGT ATT GAA AAC ATC GAT GAA GCT ATT AC - #A CTG CTT GAG CAA AAT     511    Thr Gly Ile Glu Asn Ile Asp Glu Ala Ile Th - #r Leu Leu Glu Gln Asn    #         30    - AAC TGG GAC TTG GTG GCA GCT ATT AAT GGT GT - #A ATA CCA CAG GAA AAT     559    Asn Trp Asp Leu Val Ala Ala Ile Asn Gly Va - #l Ile Pro Gln Glu Asn    #     45    - GGC ATT CTA CAA AGT GAC TTT GGA GGT GAG AC - #C ATG CCA GGA CCC ACA     607    Gly Ile Leu Gln Ser Asp Phe Gly Gly Glu Th - #r Met Pro Gly Pro Thr    # 65    - TTT GAT CCA GCA AGT CAC CCT GCT CCA GCT TC - #A ACT CCC TCT TCT TCA     655    Phe Asp Pro Ala Ser His Pro Ala Pro Ala Se - #r Thr Pro Ser Ser Ser    #                 80    - GCG TTT CGA CCT GTA ATG CCA TCC AGG CAG AT - #T GTA GAA AGG CAG CCT     703    Ala Phe Arg Pro Val Met Pro Ser Arg Gln Il - #e Val Glu Arg Gln Pro    #             95    - CGA ATG CTA GAC TTC AGA GTT GAA TAC AGA GA - #C AGA AAT GTT GAT GTG     751    Arg Met Leu Asp Phe Arg Val Glu Tyr Arg As - #p Arg Asn Val Asp Val    #       110    - GTA CTT GAA GAC AGC TGT ACT GTT GGA GAG AT - #C AAA CAG ATT CTA GAA     799    Val Leu Glu Asp Ser Cys Thr Val Gly Glu Il - #e Lys Gln Ile Leu Glu    #   125    - AAT GAG CTT CAG ATA CCT GTG CCT AAA ATG CT - #G TTA AAA GGC TGG AAG     847    Asn Glu Leu Gln Ile Pro Val Pro Lys Met Le - #u Leu Lys Gly Trp Lys    130                 1 - #35                 1 - #40                 1 -    #45    - ACT GGA GAC GTG GAA GAC AGT ACG GTC TTA AA - #A TCA CTA CAC TTG CCA     895    Thr Gly Asp Val Glu Asp Ser Thr Val Leu Ly - #s Ser Leu His Leu Pro    #               160    - AAA AAC AAC AGT CTT TAT GTC CTT ACA CCA GA - #C TTG CCA CCG CCT TCA     943    Lys Asn Asn Ser Leu Tyr Val Leu Thr Pro As - #p Leu Pro Pro Pro Ser    #           175    - TCA TCC AGC CAT GCT GGT GCC CTG CAG GAA TC - #A TTA AAT CAA AAC TTC     991    Ser Ser Ser His Ala Gly Ala Leu Gln Glu Se - #r Leu Asn Gln Asn Phe    #       190    - ATG CTG ATC ATC ACC CAC CGA GAG GTC CAG CG - #G GAG TAC AAC CTG AAC    1039    Met Leu Ile Ile Thr His Arg Glu Val Gln Ar - #g Glu Tyr Asn Leu Asn    #   205    - TTC TCA GGA AGC AGT ACC GTT CAA GAG GTA AA - #G AGA AAT GTG TAT GAC    1087    Phe Ser Gly Ser Ser Thr Val Gln Glu Val Ly - #s Arg Asn Val Tyr Asp    210                 2 - #15                 2 - #20                 2 -    #25    - CTT ACA AGC ATA CCT GTT CGA CAT CAG TTA TG - #G GAG GGC TGG CCA GCT    1135    Leu Thr Ser Ile Pro Val Arg His Gln Leu Tr - #p Glu Gly Trp Pro Ala    #               240    - TCT GCC ACC GAT GAC TCA ATG TGT CTT GCT GA - #A TCA GGC CTC TCT TAT    1183    Ser Ala Thr Asp Asp Ser Met Cys Leu Ala Gl - #u Ser Gly Leu Ser Tyr    #           255    - CCC TGC CAT CGA TTA ACT GTG GGA AGA AGA AC - #T TCA CCT GTA CAG ACC    1231    Pro Cys His Arg Leu Thr Val Gly Arg Arg Th - #r Ser Pro Val Gln Thr    #       270    - CGT GAG CAA TCA GAA GAG CAA AGC ACG GAT GT - #T CAT ATG GTT AGT GAT    1279    Arg Glu Gln Ser Glu Glu Gln Ser Thr Asp Va - #l His Met Val Ser Asp    #   285    - AGT GAT GGC GAT GAC TTT GAA GAT GCT TCA GA - #A TTT GGA GTG GAT GAC    1327    Ser Asp Gly Asp Asp Phe Glu Asp Ala Ser Gl - #u Phe Gly Val Asp Asp    290                 2 - #95                 3 - #00                 3 -    #05    - GGA GAA GTA TTT GGC ATG GCA TCA TCT ACC CT - #G AGA AAA TCT CCA ATG    1375    Gly Glu Val Phe Gly Met Ala Ser Ser Thr Le - #u Arg Lys Ser Pro Met    #               320    - ATG CCA GAA AAC GCA GAA AAT GAA GGA GAT GC - #C TTA TTA CAA TTT ACA    1423    Met Pro Glu Asn Ala Glu Asn Glu Gly Asp Al - #a Leu Leu Gln Phe Thr    #           335    - GCA GAG TTT TCT TCA AGA TAT AGT GAC TGC CA - #T CCT GTA TTT TAT ATT    1471    Ala Glu Phe Ser Ser Arg Tyr Ser Asp Cys Hi - #s Pro Val Phe Tyr Ile    #       350    - GGC TCA TTA GAA GCT GCT TTC CAA GAG GCC TT - #C TAT GTG AAA GCC CGA    1519    Gly Ser Leu Glu Ala Ala Phe Gln Glu Ala Ph - #e Tyr Val Lys Ala Arg    #   365    - GAC AGA AAA CTT CTT GCT ATC TAC CTC CAC CA - #T GAT GAA AGT GTA CTA    1567    Asp Arg Lys Leu Leu Ala Ile Tyr Leu His Hi - #s Asp Glu Ser Val Leu    370                 3 - #75                 3 - #80                 3 -    #85    - ACC AAC GTG TTC TGC TCA CAA ATG CTT TGT GC - #T GAA TCC ATT GTT TCC    1615    Thr Asn Val Phe Cys Ser Gln Met Leu Cys Al - #a Glu Ser Ile Val Ser    #               400    - TAT CTG AGT CAA AAT TTT ATA ACC TGG GCT TG - #G GAT CTG ACA AAG GAC    1663    Tyr Leu Ser Gln Asn Phe Ile Thr Trp Ala Tr - #p Asp Leu Thr Lys Asp    #           415    - ACC AAC AGA GCA AGA TTT CTG ACA ATG TGC AA - #T AGA CAC TTT GGC AGC    1711    Thr Asn Arg Ala Arg Phe Leu Thr Met Cys As - #n Arg His Phe Gly Ser    #       430    - GTT ATT GCA CAA ACT ATT CGG ACT CAA AAG AC - #A GAT CAG TTT CCA CTT    1759    Val Ile Ala Gln Thr Ile Arg Thr Gln Lys Th - #r Asp Gln Phe Pro Leu    #   445    - TTC CTG ATT ATC ATG GGA AAG CGA TCA TCT AA - #T GAA GTG TTA AAT GTG    1807    Phe Leu Ile Ile Met Gly Lys Arg Ser Ser As - #n Glu Val Leu Asn Val    450                 4 - #55                 4 - #60                 4 -    #65    - ATA CAA GGT AAT ACA ACA GTG GAT GAG TTA AT - #G ATG AGA CTC ATG GCT    1855    Ile Gln Gly Asn Thr Thr Val Asp Glu Leu Me - #t Met Arg Leu Met Ala    #               480    - GCA ATG GAG ATT TTC TCA GCT CAA CAA CAG GA - #A GAC ATT AAG GAT GAG    1903    Ala Met Glu Ile Phe Ser Ala Gln Gln Gln Gl - #u Asp Ile Lys Asp Glu    #           495    - GAT GAA CGT GAA GCC AGA GAA AAT GTG AAG AG - #A GAG CAA GAT GAG GCC    1951    Asp Glu Arg Glu Ala Arg Glu Asn Val Lys Ar - #g Glu Gln Asp Glu Ala    #       510    - TAT CGC CTT TCC CTC GAA GCC GAC AGG GCA AA - #G AGA GAA GCT CAT GAG    1999    Tyr Arg Leu Ser Leu Glu Ala Asp Arg Ala Ly - #s Arg Glu Ala His Glu    #   525    - AGA GAG ATG GCA GAA CAG TTT CGT TTG GAG CA - #G ATT CGC AAA GAA CAA    2047    Arg Glu Met Ala Glu Gln Phe Arg Leu Glu Gl - #n Ile Arg Lys Glu Gln    530                 5 - #35                 5 - #40                 5 -    #45    - GAA GAA GAA CGT GAG GCC ATC AGA CTC TCC TT - #A GAA CAA GCC CTT CCT    2095    Glu Glu Glu Arg Glu Ala Ile Arg Leu Ser Le - #u Glu Gln Ala Leu Pro    #               560    - CCA GAG CCG AAG GAA GAA AAT GCT GAG CCT GT - #T AGC AAG CTT CGG ATT    2143    Pro Glu Pro Lys Glu Glu Asn Ala Glu Pro Va - #l Ser Lys Leu Arg Ile    #           575    - CGA ACC CCC AGT GGC GAG TTC CTG GAA CGG CG - #T TTC CTG GCC AGC AAT    2191    Arg Thr Pro Ser Gly Glu Phe Leu Glu Arg Ar - #g Phe Leu Ala Ser Asn    #       590    - AAG CTC CAG ATT GTC TTT GAT TTC GTG GCT TC - #C AAG GGA TTT CCA TGG    2239    Lys Leu Gln Ile Val Phe Asp Phe Val Ala Se - #r Lys Gly Phe Pro Trp    #   605    - GAT GAA TTC AAG TTA CTG AGC ACC TTT CCT AG - #G AGA GAT GTA ACT CAG    2287    Asp Glu Phe Lys Leu Leu Ser Thr Phe Pro Ar - #g Arg Asp Val Thr Gln    610                 6 - #15                 6 - #20                 6 -    #25    - CTA GAC CCC AAT AAG TCA TTA TTG GAG GTA AA - #C TTG TTC CCT CAA GAA    2335    Leu Asp Pro Asn Lys Ser Leu Leu Glu Val As - #n Leu Phe Pro Gln Glu    #               640    - ACC CTT TTC CTT CAA GCA AAA GAG TAAACATGAC TG - #AGAGGTGG AAGCAGCCAC    2389    Thr Leu Phe Leu Gln Ala Lys Glu                645    - TCCTGACGAG CCAGCGGCAC GTGTCAAGAG ATGGGCTCCT CACCAACCCA CC - #TACCTGCT    2449    - CGTGTCACTC AGTTCAATGT CACACTTCTG CCTCTTGCAA GATTGCTGGA AA - #AAAGTAAT    2509    #             2558AAAAA AAAAAAAAAA AAACCCTGAC GTAGCTCGA    - (2) INFORMATION FOR SEQ ID NO:2:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 649 amino              (B) TYPE: amino acid              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: protein    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:    - Met Ala Ser Asn Met Asp Leu Pro Met Ile Le - #u Ala Asp Phe Gln Ala    #                 15    - Cys Thr Gly Ile Glu Asn Ile Asp Glu Ala Il - #e Thr Leu Leu Glu Gln    #             30    - Asn Asn Trp Asp Leu Val Ala Ala Ile Asn Gl - #y Val Ile Pro Gln Glu    #         45    - Asn Gly Ile Leu Gln Ser Asp Phe Gly Gly Gl - #u Thr Met Pro Gly Pro    #     60    - Thr Phe Asp Pro Ala Ser His Pro Ala Pro Al - #a Ser Thr Pro Ser Ser    # 80    - Ser Ala Phe Arg Pro Val Met Pro Ser Arg Gl - #n Ile Val Glu Arg Gln    #                 95    - Pro Arg Met Leu Asp Phe Arg Val Glu Tyr Ar - #g Asp Arg Asn Val Asp    #           110    - Val Val Leu Glu Asp Ser Cys Thr Val Gly Gl - #u Ile Lys Gln Ile Leu    #       125    - Glu Asn Glu Leu Gln Ile Pro Val Pro Lys Me - #t Leu Leu Lys Gly Trp    #   140    - Lys Thr Gly Asp Val Glu Asp Ser Thr Val Le - #u Lys Ser Leu His Leu    145                 1 - #50                 1 - #55                 1 -    #60    - Pro Lys Asn Asn Ser Leu Tyr Val Leu Thr Pr - #o Asp Leu Pro Pro Pro    #               175    - Ser Ser Ser Ser His Ala Gly Ala Leu Gln Gl - #u Ser Leu Asn Gln Asn    #           190    - Phe Met Leu Ile Ile Thr His Arg Glu Val Gl - #n Arg Glu Tyr Asn Leu    #       205    - Asn Phe Ser Gly Ser Ser Thr Val Gln Glu Va - #l Lys Arg Asn Val Tyr    #   220    - Asp Leu Thr Ser Ile Pro Val Arg His Gln Le - #u Trp Glu Gly Trp Pro    225                 2 - #30                 2 - #35                 2 -    #40    - Ala Ser Ala Thr Asp Asp Ser Met Cys Leu Al - #a Glu Ser Gly Leu Ser    #               255    - Tyr Pro Cys His Arg Leu Thr Val Gly Arg Ar - #g Thr Ser Pro Val Gln    #           270    - Thr Arg Glu Gln Ser Glu Glu Gln Ser Thr As - #p Val His Met Val Ser    #       285    - Asp Ser Asp Gly Asp Asp Phe Glu Asp Ala Se - #r Glu Phe Gly Val Asp    #   300    - Asp Gly Glu Val Phe Gly Met Ala Ser Ser Th - #r Leu Arg Lys Ser Pro    305                 3 - #10                 3 - #15                 3 -    #20    - Met Met Pro Glu Asn Ala Glu Asn Glu Gly As - #p Ala Leu Leu Gln Phe    #               335    - Thr Ala Glu Phe Ser Ser Arg Tyr Ser Asp Cy - #s His Pro Val Phe Tyr    #           350    - Ile Gly Ser Leu Glu Ala Ala Phe Gln Glu Al - #a Phe Tyr Val Lys Ala    #       365    - Arg Asp Arg Lys Leu Leu Ala Ile Tyr Leu Hi - #s His Asp Glu Ser Val    #   380    - Leu Thr Asn Val Phe Cys Ser Gln Met Leu Cy - #s Ala Glu Ser Ile Val    385                 3 - #90                 3 - #95                 4 -    #00    - Ser Tyr Leu Ser Gln Asn Phe Ile Thr Trp Al - #a Trp Asp Leu Thr Lys    #               415    - Asp Thr Asn Arg Ala Arg Phe Leu Thr Met Cy - #s Asn Arg His Phe Gly    #           430    - Ser Val Ile Ala Gln Thr Ile Arg Thr Gln Ly - #s Thr Asp Gln Phe Pro    #       445    - Leu Phe Leu Ile Ile Met Gly Lys Arg Ser Se - #r Asn Glu Val Leu Asn    #   460    - Val Ile Gln Gly Asn Thr Thr Val Asp Glu Le - #u Met Met Arg Leu Met    465                 4 - #70                 4 - #75                 4 -    #80    - Ala Ala Met Glu Ile Phe Ser Ala Gln Gln Gl - #n Glu Asp Ile Lys Asp    #               495    - Glu Asp Glu Arg Glu Ala Arg Glu Asn Val Ly - #s Arg Glu Gln Asp Glu    #           510    - Ala Tyr Arg Leu Ser Leu Glu Ala Asp Arg Al - #a Lys Arg Glu Ala His    #       525    - Glu Arg Glu Met Ala Glu Gln Phe Arg Leu Gl - #u Gln Ile Arg Lys Glu    #   540    - Gln Glu Glu Glu Arg Glu Ala Ile Arg Leu Se - #r Leu Glu Gln Ala Leu    545                 5 - #50                 5 - #55                 5 -    #60    - Pro Pro Glu Pro Lys Glu Glu Asn Ala Glu Pr - #o Val Ser Lys Leu Arg    #               575    - Ile Arg Thr Pro Ser Gly Glu Phe Leu Glu Ar - #g Arg Phe Leu Ala Ser    #           590    - Asn Lys Leu Gln Ile Val Phe Asp Phe Val Al - #a Ser Lys Gly Phe Pro    #       605    - Trp Asp Glu Phe Lys Leu Leu Ser Thr Phe Pr - #o Arg Arg Asp Val Thr    #   620    - Gln Leu Asp Pro Asn Lys Ser Leu Leu Glu Va - #l Asn Leu Phe Pro Gln    625                 6 - #30                 6 - #35                 6 -    #40    - Glu Thr Leu Phe Leu Gln Ala Lys Glu                    645    - (2) INFORMATION FOR SEQ ID NO:3:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 4 amino              (B) TYPE: amino acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: peptide    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:    - Arg Ser Pro Leu    - (2) INFORMATION FOR SEQ ID NO:4:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 6 amino              (B) TYPE: amino acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: peptide    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:    - Gly Cys Arg Asn Ser Ile    1               5    __________________________________________________________________________

What is claimed is:
 1. An antibody that specifically binds to anisolated polypeptide comprising a domain of a Fas-associated factor 1(FAF1) polypeptide, said polypeptide capable of associating with acytoplasmic domain of Fas, wherein said FAF1 domain is encoded by anucleic acid sequence that comprises at least 18 nucleotides andhybridizes to the complementary nucleic acid sequence shown in SEQ IDNO:1 or to a degenerate form thereof.
 2. The antibody of claim 1,wherein the polypeptide comprises the sequence of SEQ ID NO:2.
 3. A kitcomprising an antibody of claim
 1. 4. The antibody of claim 1, which isa rabbit antiserum or a monoclonal antibody.
 5. A hybridoma capable ofproducing the monoclonal antibody of claim 4.